@article{ghaffari_riehn_2024, title={Plasmonic interferometer for nanoscale refractometry}, url={https://arxiv.org/abs/2406.06364}, DOI={10.48550/ARXIV.2406.06364}, abstractNote={We numerically study a double-slit interferometer that detects the modification of local phase and amplitude signal due to a refractive index contrast in a nanoplasmonic sensor after the plasmonic wave has been coupled to the farfield. Specifically, the sensor consists of two elongated zero-mode waveguides (ZMW) which function to launch surface plasmons as well as transmit light directly into the farfield. The surface plasmon waves are coupled to the farfield by surface gratings which cover one quarter of a bull-eye antenna, with different transmission directions. In the farfield, the coupled plasmon waves interfere with the directly transmitted reference wave, which reveals a combination of the phase and amplitude modification by the asymmetry of refractive index in the lumen of the ZMW. We report a drastic increase in sensitivity over interferometers that only utilize the directly transmitted fraction.}, publisher={arXiv}, author={Ghaffari, Abbas and Riehn, Robert}, year={2024}, month={Jun} } @misc{riehn_2024, title={Probing protein-DNA interactions and compaction in nanochannels}, volume={88}, ISSN={["1879-033X"]}, url={https://doi.org/10.1016/j.sbi.2024.102914}, DOI={10.1016/j.sbi.2024.102914}, abstractNote={DNA confined to nanofluidic channels with a cross-section from tens to hundreds of nm wide and hundreds of microns long stretches in an equilibrium process free of flow or end tethering. Because DNA is free to move along the channel axis, its extension is exquisitely sensitive to DNA-DNA interactions and the DNA persistence length, as well as the contour length. We discuss how this sensitivity has been used to probe DNA-protein interactions at physiological concentrations of both DNA and proteins.}, journal={CURRENT OPINION IN STRUCTURAL BIOLOGY}, author={Riehn, Robert}, year={2024}, month={Oct} } @article{ghaffari_kashani_do_weninger_riehn_2023, title={A nanophotonic interferometer}, volume={34}, ISSN={["1361-6528"]}, url={https://doi.org/10.1088/1361-6528/acb443}, DOI={10.1088/1361-6528/acb443}, abstractNote={Abstract The transmission of light through sub-wavelength apertures (zero-mode waveguides, ZMW) in metal films is well-explored. It introduces both an amplitude modulation as well as a phase shift to the oscillating electromagnetic field. We propose a nanophotonic interferometer by bringing two ZMW (∼100 nm diameter) in proximity and monitoring the distribution of transmitted light in the back-focal plane of collecting microscope objective (1.3 N.A.). We demonstrate that both an asymmetry induced by the binding of a quantum dot in one of the two ZMW, as well as an asymmetry in ZMW diameter yield qualitatively similar transmission patterns. We find that the complex pattern can be quantified through a scalar measure of asymmetry along the symmetry axis of the aperture pair. In a combined experimental and computational exploration of detectors with differing ZMW diameters, we find that the scalar asymmetry is a monotonous function of the diameter difference of the two apertures, and that the scalar asymmetry measure is higher if the sample is slightly displaced from the focal plane of the collecting microscope objective. An optimization of the detector geometry determined that the maximum response is achieved at an aperture separation that is comparable to the wavelength on the exit side of the sensor. For small separations of apertures, on the order of a quarter of the wavelength and less, the signal is strongly polarization dependent, while for larger separations, on the order of the wavelength or larger, the signal becomes essentially polarization-independent.}, number={18}, journal={NANOTECHNOLOGY}, author={Ghaffari, Abbas and Kashani, Somayeh and Do, Kevin and Weninger, Keith and Riehn, Robert}, year={2023}, month={Apr} } @article{liu_pan_kaur_wang_jin_detwiler_opresko_tao_wang_riehn_2023, title={Assembly path dependence of telomeric DNA compaction by TRF1, TIN2, and SA1}, volume={122}, ISSN={["1542-0086"]}, url={https://doi.org/10.1016/j.bpj.2023.04.014}, DOI={10.1016/j.bpj.2023.04.014}, abstractNote={Telomeres, complexes of DNA and proteins, protect ends of linear chromosomes. In humans, the two shelterin proteins TRF1 and TIN2, along with cohesin subunit SA1, were proposed to mediate telomere cohesion. Although the ability of the TRF1-TIN2 and TRF1-SA1 systems to compact telomeric DNA by DNA-DNA bridging has been reported, the function of the full ternary TRF1-TIN2-SA1 system has not been explored in detail. Here, we quantify the compaction of nanochannel-stretched DNA by the ternary system, as well as its constituents, and obtain estimates of the relative impact of its constituents and their interactions. We find that TRF1, TIN2, and SA1 work synergistically to cause a compaction of the DNA substrate, and that maximal compaction occurs if all three proteins are present. By altering the sequence with which DNA substrates are exposed to proteins, we establish that compaction by TRF1 and TIN2 can proceed through binding of TRF1 to DNA, followed by compaction as TIN2 recognizes the previously bound TRF1. We further establish that SA1 alone can also lead to a compaction, and that compaction in a combined system of all three proteins can be understood as an additive effect of TRF1-TIN2 and SA1-based compaction. Atomic force microscopy of intermolecular aggregation confirms that a combination of TRF1, TIN2, and SA1 together drive strong intermolecular aggregation as it would be required during chromosome cohesion.}, number={10}, journal={BIOPHYSICAL JOURNAL}, author={Liu, Ming and Pan, Hai and Kaur, Parminder and Wang, Lucia J. and Jin, Miao and Detwiler, Ariana C. and Opresko, Patricia L. and Tao, Yizhi Jane and Wang, Hong and Riehn, Robert}, year={2023}, month={May}, pages={1822–1832} } @article{ghaffari_do_kashani_weninger_riehn_2022, title={A Nanophotonic Interferometer for small particle detection}, volume={12223}, ISBN={["978-1-5106-5430-3"]}, ISSN={["1996-756X"]}, DOI={10.1117/12.2634318}, abstractNote={The transmission of light through sub-wavelength apertures (zero-mode waveguides, ZMW) in metal films is wellexplored. It introduces both an amplitude modulation as well as a phase shift to the transmitted oscillating electromagnetic field. We propose a nanophotonic interferometer by bringing two zero-mode waveguides in proximity and monitoring the distribution of light in the back-focal plane of the collecting microscope objective. We demonstrate that both an asymmetry induced by the binding of a quantum dot in one of the two ZMW, as well as a asymmetry in ZMW diameter yield qualitatively similar deflection patterns. Using ZMW pairs with diameter asymmetries, we find that the complex pattern of the transmitted light can be quantified through a scalar measure of asymmetry along the symmetry axis of the aperture pair. We find that this scalar asymmetry is a monotonous function of the diameter difference of the two apertures.}, journal={INTERFEROMETRY XXI}, author={Ghaffari, A. and Do, K. and Kashani, S. and Weninger, K. and Riehn, Robert}, year={2022} } @article{leighton_irvin_kaur_liu_you_bhattaram_piehler_riehn_wang_pan_et al._2022, title={Densely methylated DNA traps Methyl-CpG-binding domain protein 2 but permits free diffusion by Methyl-CpG-binding domain protein 3}, volume={298}, ISSN={["1083-351X"]}, url={https://doi.org/10.1016/j.jbc.2022.102428}, DOI={10.1016/j.jbc.2022.102428}, abstractNote={The methyl-CpG–binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status. The methyl-CpG–binding domain 2 and 3 proteins (MBD2 and MBD3) provide structural and DNA-binding function for the Nucleosome Remodeling and Deacetylase (NuRD) complex. The two proteins form distinct NuRD complexes and show different binding affinity and selectivity for methylated DNA. Previous studies have shown that MBD2 binds with high affinity and selectivity for a single methylated CpG dinucleotide while MBD3 does not. However, the NuRD complex functions in regions of the genome that contain many CpG dinucleotides (CpG islands). Therefore, in this work, we investigate the binding and diffusion of MBD2 and MBD3 on more biologically relevant DNA templates that contain a large CpG island or limited CpG sites. Using a combination of single-molecule and biophysical analyses, we show that both MBD2 and MBD3 diffuse freely and rapidly across unmethylated CpG-rich DNA. In contrast, we found methylation of large CpG islands traps MBD2 leading to stable and apparently static binding on the CpG island while MBD3 continues to diffuse freely. In addition, we demonstrate both proteins bend DNA, which is augmented by methylation. Together, these studies support a model in which MBD2-NuRD strongly localizes to and compacts methylated CpG islands while MBD3-NuRD can freely mobilize nucleosomes independent of methylation status. The methyl-CpG–binding domain (MBD) family of proteins binds methylated DNA through a conserved domain that recognizes the symmetrically related methylcytosines in a cytosine-guanosine dinucleotide (CpG) (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar). The structure of this domain bound to a single methylated CpG (mCpG) site has been determined for most members of the MBD family (2Ohki I. Shimotake N. Fujita N. Jee J. Ikegami T. Nakao M. et al.Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA.Cell. 2001; 105: 487-497Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 3Ho K.L. McNae I.W. Schmiedeberg L. Klose R.J. Bird A.P. Walkinshaw M.D. MeCP2 binding to DNA depends upon hydration at methyl-CpG.Mol. Cell. 2008; 29: 525-531Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 4Scarsdale J.N. Webb H.D. Ginder G.D. Williams Jr., D.C. Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence.Nucleic Acids Res. 2011; 39: 6741-6752Crossref PubMed Scopus (80) Google Scholar, 5Walavalkar N.M. Cramer J.M. Buchwald W.A. Scarsdale J.N. Williams Jr., D.C. Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches.Nucleic Acids Res. 2014; 42: 11218-11232Crossref PubMed Scopus (24) Google Scholar, 6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 7Otani J. Arita K. Kato T. Kinoshita M. Kimura H. Suetake I. et al.Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4.J. Biol. Chem. 2013; 288: 6351-6362Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Liu K. Xu C. Lei M. Yang A. Loppnau P. 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Links between DNA methylation and nucleosome occupancy in the human genome.Epigenetics Chromatin. 2017; 10: 18Crossref PubMed Scopus (41) Google Scholar, 13Collings C.K. Waddell P.J. Anderson J.N. Effects of DNA methylation on nucleosome stability.Nucleic Acids Res. 2013; 41: 2918-2931Crossref PubMed Scopus (68) Google Scholar, 14Doi A. Park I.H. Wen B. Murakami P. Aryee M.J. Irizarry R. et al.Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts.Nat. Genet. 2009; 41: 1350-1353Crossref PubMed Scopus (965) Google Scholar). Furthermore, methylation of CpG islands in promoters and enhancers correlates with nucleosome occupancy, chromatin compaction, and associated gene silencing. Hence, we have investigated how MBD proteins bind and diffuse along these CpG islands to better understand the functional consequences in a more biologically relevant context. In the current work, we focus on the structure and dynamics of the MBD2 and MBD3 proteins. These two highly homologous proteins arose from a duplication of the ancestral MBD present across the animal kingdom (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar, 15Cramer J.M. Pohlmann D. Gomez F. Mark L. Kornegay B. Hall C. et al.Methylation specific targeting of a chromatin remodeling complex from sponges to humans.Sci. Rep. 2017; 740674Crossref Scopus (20) Google Scholar). They contribute to the structure and function of the Nucleosome Remodeling and Deacetylase (NuRD) (16Le Guezennec X. Vermeulen M. Brinkman A.B. Hoeijmakers W.A. Cohen A. Lasonder E. et al.MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties.Mol. Cell. Biol. 2006; 26: 843-851Crossref PubMed Scopus (266) Google Scholar) complex that can reposition nucleosomes, deacetylate histones, and modify gene expression. The NuRD complex (17Torrado M. Low J.K.K. Silva A.P.G. Schmidberger J.W. Sana M. Sharifi Tabar M. et al.Refinement of the subunit interaction network within the nucleosome remodelling and deacetylase (NuRD) complex.FEBS J. 2017; 284: 4216-4232Crossref PubMed Scopus (39) Google Scholar, 18Allen H.F. Wade P.A. Kutateladze T.G. The NuRD architecture.Cell. Mol. Life Sci. 2013; 70: 3513-3524Crossref PubMed Scopus (126) Google Scholar, 19Zhang W. Aubert A. Gomez de Segura J.M. Karuppasamy M. Basu S. Murthy A.S. et al.The nucleosome remodeling and deacetylase complex NuRD is built from preformed catalytically active sub-modules.J. Mol. Biol. 2016; 428: 2931-2942Crossref PubMed Scopus (47) Google Scholar, 20Zhang Y. Ng H.H. Erdjument-Bromage H. Tempst P. Bird A. Reinberg D. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.Genes Dev. 1999; 13: 1924-1935Crossref PubMed Scopus (937) Google Scholar) consists of a least six additional proteins, each of which has multiple paralogs that provide histone deacetylase activity (HDAC1/2), histone binding, and chromatin remodeling function (CHD3/4), and protein–protein interactions (GATAD2A/B, RBBP4/7, MTA1/2/3, CDK2AP1). The MBD2 and MBD3 proteins form distinct NuRD complexes that appear to have unique functional roles (16Le Guezennec X. Vermeulen M. Brinkman A.B. Hoeijmakers W.A. Cohen A. Lasonder E. et al.MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties.Mol. Cell. Biol. 2006; 26: 843-851Crossref PubMed Scopus (266) Google Scholar, 21Leighton G. Williams Jr., D.C. The methyl-CpG-binding domain 2 and 3 proteins and formation of the nucleosome remodeling and deacetylase complex.J. Mol. Biol. 2020; 432: 1624-1639Crossref Scopus (18) Google Scholar, 22Yu X. Azzo A. Bilinovich S.M. Li X. Dozmorov M. Kurita R. et al.Disruption of the MBD2-NuRD complex but not MBD3-NuRD induces high level HbF expression in human adult erythroid cells.Haematologica. 2019; 104: 2361-2371Crossref PubMed Scopus (32) Google Scholar). The two proteins show different levels of selectivity for mCpGs attributable primarily to a single amino acid change from tyrosine (MBD2) to phenylalanine (MBD3) within the DNA-binding site (Fig. 1) (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar, 6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 24Saito M. Ishikawa F. The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2.J. Biol. Chem. 2002; 277: 35434-35439Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). MBD2 shows up to 100-fold selectivity for a fully mCpG dinucleotide compared to an unmethylated CpG (1Hendrich B. Bird A. Identification and characterization of a family of mammalian methyl-CpG binding proteins.Mol. Cell. Biol. 1998; 18: 6538-6547Crossref PubMed Scopus (1089) Google Scholar, 4Scarsdale J.N. Webb H.D. Ginder G.D. Williams Jr., D.C. Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence.Nucleic Acids Res. 2011; 39: 6741-6752Crossref PubMed Scopus (80) Google Scholar, 25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar, 26Hashimoto H. Liu Y. Upadhyay A.K. Chang Y. Howerton S.B. Vertino P.M. et al.Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.Nucleic Acids Res. 2012; 40: 4841-4849Crossref PubMed Scopus (356) Google Scholar). In contrast, MBD3 binds DNA with an overall much lower affinity and shows no or slight (3–5 fold) selectivity for an mCpG (6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 26Hashimoto H. Liu Y. Upadhyay A.K. Chang Y. Howerton S.B. Vertino P.M. et al.Recognition and potential mechanisms for replication and erasure of cytosine hydroxymethylation.Nucleic Acids Res. 2012; 40: 4841-4849Crossref PubMed Scopus (356) Google Scholar). Consistent with this binding difference, previous genomic localization studies found that MBD2-NuRD predominantly binds at heavily mCpG islands associated with silenced genes, whereas MBD3 localizes to methylated and unmethylated CpG islands associated with expressed genes (23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 27Menafra R. Brinkman A.B. Matarese F. Franci G. Bartels S.J. Nguyen L. et al.Genome-wide binding of MBD2 reveals strong preference for highly methylated loci.PLoS One. 2014; 9: e99603Crossref PubMed Scopus (37) Google Scholar, 28Shimbo T. Du Y. Grimm S.A. Dhasarathy A. Mav D. Shah R.R. et al.MBD3 localizes at promoters, gene bodies and enhancers of active genes.Plos Genet. 2013; 9e1004028Crossref PubMed Scopus (82) Google Scholar, 29Gunther K. Rust M. Leers J. Boettger T. Scharfe M. Jarek M. et al.Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences.Nucleic Acids Res. 2013; 41: 3010-3021Crossref PubMed Scopus (90) Google Scholar). However, recent data suggests the alternative interpretation that both MBD2 and MBD3 depend on methylation for proper localization across the genome (30Hainer S.J. McCannell K.N. Yu J. Ee L.-S. Zhu L.J. Rando O.J. et al.DNA methylation directs genomic localization of Mbd2 and Mbd3 in embryonic stem cells.Elife. 2016; 5: e21964Crossref PubMed Scopus (21) Google Scholar). Therefore, how methylation selectivity of the MBD2 and MBD3 proteins impacts this localization and function remains an open question in the field. In previous work, we used single-molecule analyses to study the behavior of the isolated MBD from MBD2 on various DNA substrates (31Pan H. Bilinovich S.M. Kaur P. Riehn R. Wang H. Williams Jr., D.C. CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level.Nucleic Acids Res. 2017; 45: 9164-9177Crossref PubMed Scopus (19) Google Scholar). Consistent with NMR and bulk biochemical studies, we found a remarkable difference in DNA bending and sliding of the MBD from MBD2 (MBD2MBD) on methylated and unmethylated DNA containing CpG islands. MBD2MBD is mostly restricted to the mCpG islands, while it freely diffuses across unmethylated CpG islands. Furthermore, we also uncovered a novel role for the intrinsically disordered region (IDR) of MBD2 in DNA bending (25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar). The DNA-bending angle induced by both the MBD and a small portion of the IDR (MBD2MBD+IDR) on unmethylated CpG-rich DNA is larger than observed for CpG-free and further increases upon binding mCpG-rich DNA. In separate structural studies of MBD3, we found that the MBD from MBD3 shows only weak selectivity for a single mCpG within a small (17 bp) dsDNA fragment (6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Based on a combination of chemical shift analyses, mutagenesis, and residual dipolar coupling measurements, we showed that MBD3 exchanges rapidly between CpG-specific and nonspecific binding modes, leading to chemical shift averaging between these two states. Hence, MBD3 recognizes an mCpG site, as evidenced by significant chemical shift changes, but does not strongly localize to this site when bound to DNA. This difference between the DNA-binding dynamics of MBD2 and MBD3 correlates with the prior localization studies that show both MBD2 and MBD3 localize to unmethylated CpG islands, while MBD2 more exclusively localizes to mCpG islands (23Baubec T. Ivanek R. Lienert F. Schubeler D. Methylation-dependent and -independent genomic targeting principles of the MBD protein family.Cell. 2013; 153: 480-492Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 27Menafra R. Brinkman A.B. Matarese F. Franci G. Bartels S.J. Nguyen L. et al.Genome-wide binding of MBD2 reveals strong preference for highly methylated loci.PLoS One. 2014; 9: e99603Crossref PubMed Scopus (37) Google Scholar, 28Shimbo T. Du Y. Grimm S.A. Dhasarathy A. Mav D. Shah R.R. et al.MBD3 localizes at promoters, gene bodies and enhancers of active genes.Plos Genet. 2013; 9e1004028Crossref PubMed Scopus (82) Google Scholar, 29Gunther K. Rust M. Leers J. Boettger T. Scharfe M. Jarek M. et al.Differential roles for MBD2 and MBD3 at methylated CpG islands, active promoters and binding to exon sequences.Nucleic Acids Res. 2013; 41: 3010-3021Crossref PubMed Scopus (90) Google Scholar). Despite these recent studies, we do not know how the remaining, largely unstructured regions of MBD2 and MBD3 influence diffusion along DNA. Furthermore, it is unclear how reduced selectivity and binding affinity of MBD3 modifies its distribution and sliding on methylated and unmethylated CpG islands, which contain many CpG sites. To address these questions, in the current studies, we use a combination of biophysical techniques, including atomic force microscopy (AFM) imaging (32Benarroch-Popivker D. Pisano S. Mendez-Bermudez A. Lototska L. Kaur P. Bauwens S. et al.TRF2-Mediated control of telomere DNA topology as a mechanism for chromosome-end protection.Mol. Cell. 2016; 61: 274-286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 33Wang H. Tessmer I. Croteau D.L. Erie D.A. Van Houten B. 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Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar), and NMR (5Walavalkar N.M. Cramer J.M. Buchwald W.A. Scarsdale J.N. Williams Jr., D.C. Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches.Nucleic Acids Res. 2014; 42: 11218-11232Crossref PubMed Scopus (24) Google Scholar, 6Cramer J.M. Scarsdale J.N. Walavalkar N.M. Buchwald W.A. Ginder G.D. Williams Jr., D.C. Probing the dynamic distribution of bound states for methylcytosine-binding domains on DNA.J. Biol. Chem. 2014; 289: 1294-1302Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) to measure the binding and sliding of MBD2 and MBD3 on methylated and unmethylated DNA substrates. In the DNA tightrope assay, DNA molecules are stretched under hydrodynamic flow inside a flow cell. Anchoring of stretched DNA between poly-L-lysine–coated silica microspheres leads to the formation of DNA tightropes at an elongation of ∼90% of the DNA contour length (Fig. 2A). The spatial resolution of the DNA tightrope assay was estimated to be 16 nm (37Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar). Uniquely, DNA tightropes created using tandemly ligated DNA allow us to directly correlate DNA-binding events with the underlying specific DNA sequences or structures such as three-stranded R-loops (31Pan H. Bilinovich S.M. Kaur P. Riehn R. Wang H. Williams Jr., D.C. CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level.Nucleic Acids Res. 2017; 45: 9164-9177Crossref PubMed Scopus (19) Google Scholar, 32Benarroch-Popivker D. Pisano S. Mendez-Bermudez A. Lototska L. Kaur P. Bauwens S. et al.TRF2-Mediated control of telomere DNA topology as a mechanism for chromosome-end protection.Mol. Cell. 2016; 61: 274-286Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 37Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. et al.TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (51) Google Scholar, 38Pan H. Jin M. Ghadiyaram A. Kaur P. Miller H.E. Ta H.M. et al.Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.Nucleic Acids Res. 2020; 48: 5639-5655Crossref PubMed Google Scholar). To study MBD proteins diffusion, we ligated linear DNA fragments to form DNA tightropes with CpG free or alternating CpG-free and CpG-rich regions (Fig. 2B and S8A). The results from our previous study revealed that the isolated MBD from MBD2, with or without a small portion of the adjacent IDR (MBD2MBD and MBD2MBD+IDR), carry out unbiased 1D diffusion on CpG-rich DNA but undergoes subdiffusion on CpG-free DNA. In contrast, both proteins stably and statically bind to mCpG regions. In this study, we purified a construct that contains almost the entire length of the MBD2b isoform plus the coiled-coil region from GATAD2A (MBD2sc) to investigate how these additional regions impact DNA binding and diffusion (Experimental Procedures, Fig. 1A). We previously found that the MBD2sc binds DNA with an approximately 100× higher affinity than MBD2MBD (25Desai M.A. Webb H.D. Sinanan L.M. Scarsdale J.N. Walavalkar N.M. Ginder G.D. et al.An intrinsically disordered region of methyl-CpG binding domain protein 2 (MBD2) recruits the histone deacetylase core of the NuRD complex.Nucleic Acids Res. 2015; 43: 3100-3113Crossref PubMed Scopus (44) Google Scholar). Consistent with these results, fluorescence anisotropy experiments showed that MBD2sc binds to DNA containing unmethylated, methylated, or no CpG site with equilibrium dissociation constants of 2.8 (±0.1 μM), 0.007 (±0.002 μM), or 9.1 (±0.4 μM), respectively (Fig. 2C). Hence, we questioned whether the additional binding affinity provided by the IDR in MBD2 would modify DNA binding and sliding. We directly addressed this question using the DNA tightrope assay. For the DNA tightrope assay, we first generated DNA substrates by tandemly ligating linear DNA fragments containing CpG-free or unmethylated CpG-free–rich sequences (Fig. 2B). Further, we conjugated His-tagged MBD2sc to streptavidin-coated quantum dots (SAv-QDs) through the multivalent chelator tris-nitrilotriacetic acid linker (Fig. 2A) (39Reichel A. Schaible D. Al Furoukh N. Cohen M. Schreiber G. Piehler J. Noncovalent, site-specific biotinylation of histidine-tagged proteins.Anal. Chem. 2007; 79: 8590-8600Crossref PubMed Scopus (47) Google Scholar). Following the formation of the DNA tightropes, we introduced QD-labeled MBD2sc into the flow cell. Analysis of MBD2sc on DNA tightropes revealed two populations (Table 1): apparently immobile throughout data acquisition and mobile molecules (Fig. 2D). To exclude that this apparently immobile population reflects aggregation, we categorized particles as a single protein or cluster based on their individual QD blinking rate (Table S1) which shows that only a small fraction comprises multiple proteins. To obtain diffusion coefficients for mobile MBD2sc on DNA tightropes, we tracked the position of MBD2sc-QDs on DNA by Gaussian fitting to kymographs (particle position versus time plots) (36Kad N.M. Wang H. Kennedy G.G. Warshaw D.M. Van Houten B. Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single- molecule imaging of quantum-dot-labeled proteins.Mol. Cell. 2010; 37: 702-713Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 40Dunn A.R. Kad N.M. Nelson S.R. Warshaw D.M. Wallace S.S. Single Qdot-labeled glycosylase molecules use a wedge amino acid to probe for lesions while scanning along DNA.Nucleic Acids Res. 2011; 39: 7487-7498Crossref PubMed Scopus (98) Google Scholar). We obtained diffusion coefficients and alpha exponents by fitting the mean square displacement (MSD) versus time. An alpha exponent of 1 indicates an unbiased random walk, and a value less than 1 indicates subdiffusion (41Saxton M.J. Single-particle tracking: The distribution of diffusion coefficients.Biophys. J. 1997; 72: 1744-1753Abstract Full Text PDF PubMed Scopus (479) Google Scholar). The diffusion coefficients displayed by MBD2sc on the CpG-free DNA tightrope were significantly slower than those on DNA tightropes containing CpG sites (CpG-free–rich) (Fig. 2E). In addition, on the CpG-free–rich tightropes, MBD2sc displayed alpha exponents close to 1 (1.0 ± 0.2), indicating largely unbiased 1D diffusion on DNA (Fig. 2F, Table 2). As compared to CpG-free–rich DNA, the alpha exponents for MBD2sc on CpG-free DNA tightropes were slightly (p < 0.001) reduced (0.8 ± 0.2, Fig. 2F, Table 2). Overall, MBD2sc displayed slightly different diffusion ranges on CpG-free DNA tightropes compared to CpG-free–rich DNA tightropes containing multiple CpG sites (Fig. S3). In summary, MBD2sc shows more rapid and extensive 1D diffusion on CpG-free–rich DNA than CpG-free sequences.Table 1Fraction of statically bound MBD2sc and MBD3sc on unmethylated- and methylated-DNA tightropesDNAMBD2scMBD3scStatic binding (%)NStatic binding (%)NCpG-free–rich23 ± 614747 ± 7123CpG-free19 ± 633354 ± 8240mCpG-free–rich96 ± 324652 ± 10345mCpG-mini90 ± 149550 ± 8158The values represent mean ± SD from 2 to 3 experiments for each data set. Open table in a new tab Table 2Diffusion coefficient of MBD2sc and MBD3sc on different DNA substratesDNAMBD2scMBD3scD (μm2/s)α exponentND (μm2/s)α exponentNCpG-free0.04 ± 0.020.8 ± 0.2990.04 ± 0.030.8 ± 0.3199CpG-free–rich0.15 ± 0.051.0 ± 0.21000.04 ± 0.010.9 ± 0.2144mCpG-free–richNA.NA.0.09 ± 0.021.0 ± 0.2174mCpG-miniNA.NA.0.05 ± 0.020.9 ± 0.170The values represent mean ± SD from 2 to 4 experiments for each data set. Open table in a new tab The values represent mean ± SD from 2 to 3 experiments for each data set. The values represent mean ± SD from 2 to 4 experiments for each data set. To evaluate how DNA methylation affects the dynamics of MBD2sc on DNA, we imaged QD-labeled MBD2sc on the CpG-free–rich DNA tightropes after methylation. Linear DNA substrates were methylated before ligation using CpG Methyltransferase (M.SssI) with SAM as a cofactor (Experimental procedures). We confirmed methylation of the linear CpG-free–rich DNA substrate by digestion with the methylation-sensitive HpaII restriction endonuclease (Fig. S1). We then tandemly ligated the mCpG-free–rich (mCpG-free-rich, Fig. 3A) and used it to form DNA tightropes between silica beads. Compared to unmethylated CpG-free–rich and CpG-free DNA, the binding density of MBD2sc increased approximately 4× on mCpG-free–rich DNA tightropes (Fig. 3B, S4A). This result is consistent with the preferential binding of MBD2sc to mCpG sites, as supported by binding affinity measurements (Fig. 2C). Notably, while the majority of MBD2sc observed on unmethylated CpG-free–rich DNA tightropes was mobile (77%), on mCpG-free–rich tightropes, the majority of the protein bound was apparently immobile (96%) throughout data acquisition (Fig. 3B and Table 1). Furthermore, the distance between adjacent proteins on the mCpG-free–rich DNA tightropes is Gaussian distributed, with the peak centered at 2.3 (±0.3 μm) (Fig. 3C). This spacing matches the calculated distances between adjacent mCpG-rich regions on DNA tightropes (Fig. 3A), considering that they are stretched to ∼90% of their contour length. Taken together, fluorescence imaging of MBD2sc on DNA tightropes establishes that MBD2sc recognizes mCpG islands through stable and apparently static binding. To evaluate whether MBD2s}, number={10}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Leighton, Gage O. and Irvin, Elizabeth Marie and Kaur, Parminder and Liu, Ming and You, Changjiang and Bhattaram, Dhruv and Piehler, Jacob and Riehn, Robert and Wang, Hong and Pan, Hai and et al.}, year={2022}, month={Oct} } @article{dangi_liu_azad_countryman_roushan_livshits_kaur_pan_shi_detwiler_et al._2022, title={Nanofluidic Device for Manipulation and Modification of DNA by Proteins}, url={http://dx.doi.org/10.1101/2022.12.29.521498}, DOI={10.1101/2022.12.29.521498}, abstractNote={Single-molecule techniques provide important details supplementing the framework obtained from traditional bulk experiments. Many cellular processes such as DNA replication, DNA repair, and telomere maintenance involve interaction among multiple proteins, their co-factors, and DNA. To investigate such interactions and to differentiate the function of each component necessitate a technique that allows the combinatorial exposure of DNA to multiple proteins and co-factors as well as manipulation of the DNA configuration. We present a nanofluidic device with the capability of active combinatorial exchange of up to three buffers in real-time and dynamic manipulation of DNA under physiologically relevant conditions. We demonstrate its utility in monitoring compaction of DNA by telomeric proteins, DNA modification by an endonuclease, and DNA loop extrusion by cohesin.}, author={Dangi, Saroj and Liu, Ming and Azad, Zubair and Countryman, Preston and Roushan, Maedeh and Livshits, Gideon I. and Kaur, Parminder and Pan, Hai and Shi, Zhubing and Detwiler, Ariana C. and et al.}, year={2022}, month={Dec} } @article{pan_kaur_barnes_detwiler_sanford_liu_xu_mahn_tang_hao_et al._2021, title={Structure, dynamics, and regulation of TRF1-TIN2-mediated trans- and cis-interactions on telomeric DNA}, volume={297}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2021.101080}, abstractNote={TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. TIN2 is a core component of the shelterin complex linking double-stranded telomeric DNA-binding proteins (TRF1 and TRF2) and single-strand overhang-binding proteins (TPP1-POT1). In vivo, the large majority of TRF1 and TRF2 exist in complexes containing TIN2 but lacking TPP1/POT1; however, the role of TRF1-TIN2 interactions in mediating interactions with telomeric DNA is unclear. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 interaction using atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that the short (TIN2S) and long (TIN2L) isoforms of TIN2 facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (six TTAGGG repeats), the majority of TRF1-mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG repeats), TIN2 forms multiprotein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last on the order of minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 and the cofactor NAD+ significantly reduced TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protected the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Furthermore, we showed that TPP1 inhibits TRF1-TIN2L-mediated DNA-DNA bridging. Our study, together with previous findings, supports a molecular model in which protein assemblies at telomeres are heterogeneous with distinct subcomplexes and full shelterin complexes playing distinct roles in telomere protection and elongation. Telomeres are nucleoprotein structures that prevent the degradation or fusion of the ends of linear chromosomes, which are threatened by at least seven distinct DNA damage response (DDR) pathways (1Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1344) Google Scholar, 2Muraki K. Nyhan K. Han L. Murnane J.P. 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Cell. 2011; 44: 647-659Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Binding of TPP1 to TIN2 is required for POT1-mediated telomere protection (34Frescas D. de Lange T. Binding of TPP1 protein to TIN2 protein is required for POT1a,b protein-mediated telomere protection.J. Biol. Chem. 2014; 289: 24180-24187Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). As an integral component of the "TIN2/TPP1/POT1 processivity complex," TIN2 functions together with TPP1/POT1 to stimulate telomerase processivity (35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar). Furthermore, TIN2 directly interacts with the cohesin subunit SA1 and plays a key role in a distinct SA1-TRF1-TIN2-mediated sister telomere cohesion pathway that is largely independent of the cohesin ring subunits (8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 36Canudas S. Smith S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells.J. Cell Biol. 2009; 187: 165-173Crossref PubMed Scopus (122) Google Scholar). Binding of TRF1-TIN2 to telomeres is regulated by the poly(ADP-ribose) polymerase Tankyrase 1 (37Smith S. Giriat I. Schmitt A. de Lange T. Tankyrase, a poly(ADP-ribose) polymerase at human telomeres.Science. 1998; 282: 1484-1487Crossref PubMed Scopus (891) Google Scholar). ADP-ribosylation of TRF1 by Tankyrase 1 reduces its binding to telomeric DNA in vitro, and the depletion of Tankyrase 1 using siRNA leads to mitotic arrest and persistent telomere cohesion that can be rescued by depletion of TIN2 (8Canudas S. Houghtaling B.R. Kim J.Y. Dynek J.N. Chang W.G. Smith S. Protein requirements for sister telomere association in human cells.EMBO J. 2007; 26: 4867-4878Crossref PubMed Scopus (80) Google Scholar, 36Canudas S. Smith S. Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells.J. Cell Biol. 2009; 187: 165-173Crossref PubMed Scopus (122) Google Scholar, 38Dynek J.N. Smith S. Resolution of sister telomere association is required for progression through mitosis.Science. 2004; 304: 97-100Crossref PubMed Scopus (216) Google Scholar). Three distinct TIN2 isoforms have been identified in human cell lines (35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar, 39Kaminker P.G. Kim S.H. Desprez P.Y. Campisi J. A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar, 40Smith S. The long and short of it: A new isoform of TIN2 in the nuclear matrix.Cell Cycle. 2009; 8: 797-798Crossref PubMed Scopus (2) Google Scholar) that include TIN2S (354 AAs), TIN2L (451 AAs), and TIN2M (TIN2 medium, 420 AAs). TIN2S, TIN2L, and TIN2M share the same TRF1, TRF2, and TPP1-binding domains and localize to telomeres (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar, 35Pike A.M. Strong M.A. Ouyang J.P.T. Greider C.W. TIN2 functions with TPP1/POT1 to stimulate telomerase processivity.Mol. Cell. Biol. 2019; 39e00593-18Crossref PubMed Scopus (22) Google Scholar, 39Kaminker P.G. Kim S.H. Desprez P.Y. Campisi J. A novel form of the telomere-associated protein TIN2 localizes to the nuclear matrix.Cell Cycle. 2009; 8: 931-939Crossref PubMed Scopus (35) Google Scholar). Consistent with its key role in telomere maintenance, germline inactivation of TIN2 in mice is embryonic lethal (41Chiang Y.J. Kim S.H. Tessarollo L. Campisi J. Hodes R.J. Telomere-associated protein TIN2 is essential for early embryonic development through a telomerase-independent pathway.Mol. Cell. Biol. 2004; 24: 6631-6634Crossref PubMed Scopus (61) Google Scholar). Removal of TIN2 leads to the formation of telomere dysfunction-induced foci (TIFs). Importantly, clinical studies further highlight the biological significance of TIN2 in telomere protection (42Savage S.A. Giri N. Baerlocher G.M. Orr N. Lansdorp P.M. Alter B.P. TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita.Am. J. Hum. Genet. 2008; 82: 501-509Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 43Walne A.J. Vulliamy T. Beswick R. Kirwan M. Dokal I. TINF2 mutations result in very short telomeres: Analysis of a large cohort of patients with dyskeratosis congenita and related bone marrow failure syndromes.Blood. 2008; 112: 3594-3600Crossref PubMed Scopus (227) Google Scholar). TINF2, which encodes TIN2, is the second most frequently mutated gene in the telomere elongation and protection disorder dyskeratosis congenita (DKC). DKC-associated TIN2 mutations are most frequently de novo and cluster at a highly conserved region near the end of its TRF1-binding domain. Two decades of research since the first discovery of TIN2 have shed light on protein-interaction networks around TIN2 and its multifaceted roles in telomere maintenance. However, since TIN2 itself does not directly bind to DNA and instead serves as a "mediator/enhancer" for shelterin and telomerase activities, defining TIN2's distinct function at the molecular level has been challenging. The bottleneck for studying TIN2 lies in the fact that results from bulk biochemical assays do not fully reveal the heterogeneity and dynamics of the protein–protein and protein–DNA interactions. Furthermore, cell-based assays only provide information on the outcomes from downstream effectors after the knocking down of TIN2 that also removes TRF1 and TRF2 from telomeres. These approaches do not allow us to investigate the molecular structures and dynamics in which TIN2 directly participates. In vivo, the amount of TIN2 is sufficient for binding every TRF1 and TRF2 molecule (44Takai K.K. Hooper S. Blackwood S. Gandhi R. de Lange T. In vivo stoichiometry of shelterin components.J. Biol. Chem. 2010; 285: 1457-1467Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), while TPP1 and POT1 are ~10-fold less than TRF1 and TIN2. Thus, it is important to study the DNA-binding properties of TRF1-TIN2 complexes. To fill this important knowledge gap, we applied complementary single-molecule imaging platforms, including atomic force microscopy (AFM) (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 46Wang H. Nora G.J. Ghodke H. Opresko P.L. Single molecule studies of physiologically relevant telomeric tails reveal POT1 mechanism for promoting G-quadruplex unfolding.J. Biol. Chem. 2011; 286: 7479-7489Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar), total internal reflection fluorescence microscopy (TIRFM) (48Erie D.A. Weninger K.R. Single molecule studies of DNA mismatch repair.DNA Repair. 2014; 20: 71-81Crossref PubMed Scopus (46) Google Scholar), and the DNA tightrope assay to monitor TRF1-TIN2-mediated DNA compaction and DNA-DNA bridging (49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar, 50Countryman P. Fan Y. Gorthi A. Pan H. Strickland J. Kaur P. Wang X. Lin J. Lei X. White C. You C. Wirth N. Tessmer I. Piehler J. Riehn R. et al.Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates.J. Biol. Chem. 2018; 293: 1054-1069Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 51Pan H. Jin M. Ghadiyaram A. Kaur P. Miller H.E. Ta H.M. Liu M. Fan Y. Mahn C. Gorthi A. You C. Piehler J. Riehn R. Bishop A.J.R. Tao Y.J. et al.Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA.Nucleic Acids Res. 2020; 48: 5639-5655Crossref PubMed Google Scholar). Through using DNA substrates on different length scales (6 and 270 TAAGGG repeats), these imaging platforms provide complementary results demonstrating that both TIN2S and TIN2L facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. In some cases, TRF1-TIN2 is capable of mediating the bridging of multiple copies of telomeric DNA fragments. Importantly, our results demonstrate that TIN2 protects the disassembly of TRF1-TIN2-mediated DNA-DNA bridging by Tankyrase 1. In addition, the N-terminal domain of TPP1 inhibits TRF1-TIN2-mediated DNA-DNA bridging. In summary, this study uncovered the unique biophysical function of TIN2 as a telomeric architectural protein, acting together with TRF1 to mediate interactions between distant telomeric sequences. Tankyrase 1 and TPP1 regulate TRF1-TIN2-mediated DNA-DNA bridging. Furthermore, this work establishes a unique combination of single-molecule imaging platforms for future examination of TIN2 disease variants and provides a new direction for investigating molecular mechanisms underlying diverse TIN2 functions. A previous study suggested that TIN2 modulates the bridging of telomeric DNA by TRF1 (31Kim S.H. Han S. You Y.H. Chen D.J. Campisi J. The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro.EMBO Rep. 2003; 4: 685-691Crossref PubMed Scopus (42) Google Scholar). However, the bulk biochemical assays using short telomeric DNA (six telomeric repeats) did not provide information regarding the structure and dynamics of the TRF1-TIN2-DNA complex. To investigate the molecular function of TIN2, we applied AFM imaging to investigate how TIN2 affects the telomeric DNA-DNA pairing mediated by TRF1 at the single-molecule level on longer telomeric DNA substrates (270 TTAGGG repeats). We purified TRF1 (Fig. S1A) and obtained TIN2S (1–354 amino acids, 39.4 kDa) and TIN2L (1–451 amino acids, 50.0 kDa) proteins purified from insect cells (Fig. 1A and Fig. S1D). Previously, we established an AFM imaging-based calibration method to investigate the oligomeric states and protein–protein interactions by correlating AFM volumes of proteins and their molecular weights (45Yang Y. Wang H. Erie D.A. Quantitative characterization of biomolecular assemblies and interactions using atomic force microscopy.Methods. 2003; 29: 175-187Crossref PubMed Scopus (88) Google Scholar, 47Kaur P. Wu D. Lin J. Countryman P. Bradford K.C. Erie D.A. Riehn R. Opresko P.L. Wang H. Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2.Sci. Rep. 2016; 6: 20513Crossref PubMed Scopus (20) Google Scholar, 52Wang H. Yang Y. Erie D.A. Characterization of protein-protein interactions using atomic force microscopy.in: Schuck P. Protein Interactions Biophysical approaches for the Study of Complex Reversible Systems. Springer Science+Business Media, LLC, Berlin, Germany2007: 39-78Crossref Google Scholar). AFM volumes of TRF1 alone in solution showed two distinct peaks, which were consistent with TRF1 monomers (51 KDa) and dimers (102 KDa, Fig. S1B). In addition, based on the population of TRF1 under the monomer and dimer peaks (53Wang H. DellaVecchia M.J. Skorvaga M. Croteau D.L. Erie D.A. Van Houten B. UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.J. Biol. Chem. 2006; 281: 15227-15237Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), the estimated TRF1 dimer equilibrium dissociation constant (Kd) is 18.4 nM (Fig. S1C). Meanwhile, AFM volumes of purified TIN2S at 41.3 nm3 (±28.3 nm3) and TIN2L at 41.9 nm3 (±12.8 nm3) were consistent with the notion that TIN2 does not interact with itself (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), and TIN2 exists in a monomeric state in solution (Fig. S1D). Furthermore, we conducted size-exclusive chromatography using TRF1 and TIN2S and confirmed the presence of TRF1 dimers, TIN2 monomers, as well as the interaction between TRF1 and TIN2S in solution (Fig. S2). To further validate the activities of TIN2, we used electrophoresis mobility shift assays (EMSAs) to verify the interaction of TIN2 with TRF1 on a double-stranded telomeric DNA substrate (48 bp containing three TTAGGG repeats, Fig. S3, A–C). Consistent with previous studies (23Kim S.H. Kaminker P. Campisi J. TIN2, a new regulator of telomere length in human cells.Nat. Genet. 1999; 23: 405-412Crossref PubMed Scopus (418) Google Scholar), EMSA experiments showed that TIN2S and TIN2L did not directly bind to telomeric dsDNA (Fig. S3A). Both TRF1-TIN2S and TRF1-TIN2L induced a clear supershift of the telomeric DNA substrate compared with TRF1 alone (Complex III in Fig. S3, B and C), indicating the formation of stable TRF1-TIN2-telomeric DNA complexes. Next, to study TRF1-TIN2 DNA binding at the single-molecule level, we used the linear DNA substrate (5.4 kb) that contains 1.6 kb (270 TTAGGG) telomeric repeats in the middle region that is 35%–50% from DNA ends (T270 DNA, Experimental procedures, Fig. 1A) (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar). Previously, AFM and electron microscopy imaging–based studies established that TRF1 specifically binds to the telomeric region and mediates DNA-DNA pairing (21Lin J. Countryman P. Buncher N. Kaur P. E L. Zhang Y. Gibson G. You C. Watkins S.C. Piehler J. Opresko P.L. Kad N.M. Wang H. TRF1 and TRF2 use different mechanisms to find telomeric DNA but share a novel mechanism to search for protein partners at telomeres.Nucleic Acids Res. 2014; 42: 2493-2504Crossref PubMed Scopus (44) Google Scholar, 22Bianchi A. Stansel R.M. Fairall L. Griffith J.D. Rhodes D. de Lange T. TRF1 binds a bipartite telomeric site with extreme spatial flexibility.EMBO J. 1999; 18: 5735-5744Crossref PubMed Scopus (163) Google Scholar, 49Lin J. Countryman P. Chen H. Pan H. Fan Y. Jiang Y. Kaur P. Miao W. Gurgel G. You C. Piehler J. Kad N.M. Riehn R. Opresko P.L. Smith S. et al.Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing.Nucleic Acids Res. 2016; 44: 6363-6376Crossref PubMed Scopus (18) Google Scholar). To study the function of TIN2, we preincubated TRF1 without or with TIN2 (either TIN2S or TIN2L), followed by the addition}, number={3}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Pan, Hai and Kaur, Parminder and Barnes, Ryan and Detwiler, Ariana C. and Sanford, Samantha Lynn and Liu, Ming and Xu, Pengning and Mahn, Chelsea and Tang, Qingyu and Hao, Pengyu and et al.}, year={2021}, month={Sep} } @article{kaur_barnes_pan_detwiler_liu_mahn_hall_messenger_you_piehler_et al._2021, title={TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA}, volume={49}, ISSN={["1362-4962"]}, url={https://doi.org/10.1093/nar/gkab1142}, DOI={10.1093/nar/gkab1142}, abstractNote={Abstract The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2–TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein–DNA structures, and monitoring of DNA–DNA and DNA–RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA–dsDNA, dsDNA–ssDNA and dsDNA–ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.}, number={22}, journal={NUCLEIC ACIDS RESEARCH}, publisher={Oxford University Press (OUP)}, author={Kaur, Parminder and Barnes, Ryan and Pan, Hai and Detwiler, Ariana C. and Liu, Ming and Mahn, Chelsea and Hall, Jonathan and Messenger, Zach and You, Changjiang and Piehler, Jacob and et al.}, year={2021}, month={Dec}, pages={13000–13018} } @article{pan_jin_ghadiyaram_kaur_miller_ta_liu_fan_mahn_gorthi_et al._2020, title={Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA}, volume={48}, ISSN={["1362-4962"]}, url={http://dx.doi.org/10.1093/nar/gkaa284}, DOI={10.1093/nar/gkaa284}, abstractNote={Abstract Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.}, number={10}, journal={NUCLEIC ACIDS RESEARCH}, publisher={Oxford University Press (OUP)}, author={Pan, Hai and Jin, Miao and Ghadiyaram, Ashwin and Kaur, Parminder and Miller, Henry E. and Ta, Hai Minh and Liu, Ming and Fan, Yanlin and Mahn, Chelsea and Gorthi, Aparna and et al.}, year={2020}, month={Jun}, pages={5639–5655} } @article{liu_movahed_dangi_pan_kaur_bilinovich_faison_leighton_wang_williams_et al._2020, title={DNA looping by two 5-methylcytosine-binding proteins quantified using nanofluidic devices}, volume={13}, ISSN={["1756-8935"]}, DOI={10.1186/s13072-020-00339-7}, abstractNote={Abstract Background MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA–protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins. Results We demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), we determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops. Conclusions Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosine-binding proteins can vary drastically between strong compaction of DNA and no discernable large-scale impact of protein binding. We demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping. }, number={1}, journal={EPIGENETICS & CHROMATIN}, author={Liu, Ming and Movahed, Saeid and Dangi, Saroj and Pan, Hai and Kaur, Parminder and Bilinovich, Stephanie M. and Faison, Edgar M. and Leighton, Gage O. and Wang, Hong and Williams, David C., Jr. and et al.}, year={2020}, month={Mar} } @article{wang_ghadiyaram_pan_fan_kaur_gorthi_riehn_bishop_jane tao_2019, title={Cohesin SA2 and EWSR1 in R-Loop Regulation}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.2723}, DOI={10.1016/J.BPJ.2018.11.2723}, abstractNote={R-loops are three-stranded nucleic acid structures consisting of an RNA-DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. R-loops collectively occupy up to 5% of the mammalian genome, which occur at conserved hotspot including promoter and terminator regions of poly(A) dependent genes. R-loops are proposed to be the "double-edged sword" that functions as powerful regulators of gene expression and induces genome instability if it is un-regulated. Despite the importance of R-loops in a wide range of biological pathways, proteins that mediate R-loop dependent cellular function are not fully understood. Recently The Bishop group showed that Ewing sarcoma cells displayed a higher level of R-loops compared to control cell lines. EWSR1 depletion induces R-loop accumulation. In addition, the second most common mutation in Ewing sarcoma cells was found in cohesin SA2. These observations raise the important question of whether EWSR1 as well as cohesin SA1 and SA2 subunits are sensors of R-loops that regulate R-loop processing. Using atomic force microscopy (AFM) and bulk fluorescence anisotropy, we tested the hypothesis that cohesin SA1, SA2, and EWSR1 directly bind to R-loops. We observed that cohesin SA1/SA2, and EWSR1 specifically recognize R-loops. These observations suggest direct roles of cohesin SA1/SA2 and EWSR1 in R-loop regulation and how the loss of SA2 in Ewing sarcoma may provide some benefit to these cancers.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Wang, Hong and Ghadiyaram, Ashwin and Pan, Hai and Fan, Yanlin and Kaur, Parminder and Gorthi, Aparna and Riehn, Robert and Bishop, Alexander J.R. and Jane Tao, Yizhi}, year={2019}, month={Feb}, pages={505a} } @article{movahed_azad_dangi_riehn_2019, title={Direct observation of confinement-induced diffusophoresis}, volume={7}, url={https://doi.org/10.1088/1361-6528/ab31f7}, DOI={10.1088/1361-6528/ab31f7}, abstractNote={Nanofluidic devices have channel dimensions which come to within one order of magnitude of the Debye length of common aqueous solutions. Conventionally, external driving is used to create concentration polarization of ions and biomolecules in nanofluidic devices. Here we show that long-range ionic strength gradients intrinsic to all nanofluidic devices, even at equilibrium, also drive a drift of macromolecules. To demonstrate the effect, we confine long DNA to straight nanochannels of constant, rectangular cross-section (100 × 100 nm2) which are connected to large microfluidic reservoirs. The motion of DNA is observed in absence of any driving. We find that at low ionic strengths, molecules in nanochannels migrate toward the nano-micro interface, while they are undergoing purely diffusive motion in high salt. Using numerical models, we demonstrate that the motion is consistent with the ionic strength gradient at the micro-nano interface even at equilibrium, and that the dominant cause of the drift is diffusophoresis.}, journal={Nanotechnology}, publisher={IOP Publishing}, author={Movahed, Saeid and Azad, Zubair and Dangi, Saroj and Riehn, Robert}, year={2019}, month={Oct} } @article{kaur_barnes_pan_opresko_riehn_wang_2019, title={Single-Molecule Study of TRF2 Mediated DNA Compaction using Physiologically Relevant Long Telomeric DNA}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.2725}, DOI={10.1016/J.BPJ.2018.11.2725}, abstractNote={Telomeres are nucleoprotein structures that prevent the degradation or fusion of the ends of linear chromosomes by shielding them from activating DNA damage response (DDR) and DNA double-strand break (DSB) repair. Human telomeres contain ∼2 to 20 kb of TTAGGG repeats and a G-rich 3' overhang. A specialized six-protein shelterin complex, including TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, binds and prevents telomeres from being falsely recognized as double strand breaks, and regulates telomerase and DNA repair protein access. Extensive telomere shortening or dramatic telomere loss due to DNA damage causes chromosome ends to be recognized as DNA breaks, which triggers cell senescence and aging-related pathologies. Human telomeric DNA is arranged into T-loops, in which the 3' single-stranded overhang invades the upstream double-stranded region. A recent study suggests that T-loops at telomeres function as conformational switches that regulate ATM activation [1]. Further, it was also shown that TRF2 mediated DNA compaction drives T-loop formation [2]. While it has been shown that TRF2 protein is required for the T-loop formation, essential information regarding the dynamics of its formation still remains unknown. Moreover, most of the previous studies were done using short telomeric DNA sequences ∼ 20 kb. To elucidate the dynamics of TRF2 mediated DNA compaction and T-loop formation, we used single molecule methods which frequently require long DNA substrates. We established a new method for extracting and purifying long telomeric DNA from mouse liver cells using Region specific Extraction (RSE) [3].}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Kaur, Parminder and Barnes, Ryan and Pan, Hai and Opresko, Patricia and Riehn, Robert and Wang, Hong}, year={2019}, month={Feb}, pages={505a} } @article{pan_dangi_kaur_hao_weninger_riehn_opresko_wang_2019, title={TIN2 is an Architectural Protein Stabilizing TRF1 at Telomere}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.1168}, DOI={10.1016/J.BPJ.2018.11.1168}, abstractNote={Telomeres, consisting of duplex TTAGGG repeats and associating with protein complexes at chromosome ends, play a crucial role in maintaining the stability of chromosomes. The protein complex - shelterin contains six subunits (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1), which bind to telomeres and protect the chromosome ends from DNA repairing, and recruit telomerase when chromosome gets shortened abnormally. Among these six subunits, TIN2 has no affinity to either double-strand or single-strand DNA. However, it is a core component bridging the double-strand DNA binding proteins (TRF1 and TRF2) to single-strand DNA binding protein complex (TPP1-POT1). Loss of TRF1 or TRF2 binding domain in TIN2 can trigger DNA damage response. Moreover, TIN2 without the TPP1 binding domain is capable of fully supporting the stabilization of TRF1 and TRF2/RAP1. Despite the significance of TIN2 in telomere maintenance, the mechanism underlying TIN2 remains elusive. To understand how TIN2 affects TRF1 binding dynamics and how TIN2 influences with TRF1-mediated telomeric DNA pairing, we conducted Atomic Force Microscopy (AFM) and used nanochannels confined DNA to study the telomeric DNA conformation upon TRF1 and TIN2 binding. We observed high-order protein-DNA complexes formation indicating TIN2 facilitates TRF1 accumulation on telomeric DNA. We also carried out single molecule fluorescence technique to investigate how TIN2 affects TRF1 binding dynamics on telomeric DNA sequences. Our results show that TIN2 can accelerate DNA-DNA pairing and stabilize TRF1 on telomeric DNA.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Pan, Hai and Dangi, Saroj and Kaur, Parminder and Hao, Pengyu and Weninger, Keith and Riehn, Robert and Opresko, Patricia and Wang, Hong}, year={2019}, month={Feb}, pages={211a–212a} } @article{li_lim_puretzky_riehn_hallen_2018, title={DNA Methylation Detection Using Resonance and Nanobowtie-Antenna-Enhanced Raman Spectroscopy}, volume={114}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2018.04.021}, abstractNote={We show that DNA carrying 5-methylcytosine modifications or methylated DNA (m-DNA) can be distinguished from DNA with unmodified cytosine by Raman spectroscopy enhanced by both a bowtie nanoantenna and excitation resonance. In particular, m-DNA can be identified by a peak near 1000 cm−1 and changes in the Raman peaks in the 1200–1700 cm−1 band that are enhanced by the ring-absorption resonance. The identification is robust to the use of resonance Raman and nanoantenna excitation used to obtain significant signal improvement. The primary differences are three additional Raman peaks with methylation at 1014, 1239, and 1639 cm−1 and spectral intensity inversion at 1324 (C5=C6) and 1473 cm−1 (C4=N3) in m-DNA compared to that of DNA with unmodified cytosine. We attribute this to the proximity of the methyl group to the antenna, which brings the (C5=C6) mode closer to experiencing a stronger near-field enhancement. We also show distinct Raman spectral features attributed to the transition of DNA from a hydrated state, when dissolved, to a dried/denatured state. We observe a general broadening of the larger lines and a transfer of spectral weight from the ∼1470 cm−1 vibration to the two higher-energy lines of the dried m-DNA solution. We attribute the new spectral characteristics to DNA softening under high salt conditions and find that the m-DNA is still distinguishable via the ∼1000 cm−1 peak and distribution of the signal in the 1200–1700 cm−1 band. The nanoantenna gain exceeds 20,000, whereas the real signal ratio is much less because of a low average enhanced region occupancy even with these relatively high DNA concentrations. It is improved when fixed DNA in a salt crystal lies near the nanoantenna. The Raman resonance gain profile is consistent with A-term expectations, and the resonance is found at ∼259 nm excitation wavelength.}, number={11}, journal={BIOPHYSICAL JOURNAL}, author={Li, Ling and Lim, Shuang Fang and Puretzky, Alexander and Riehn, Robert and Hallen, Hans D.}, year={2018}, month={Jun}, pages={2498–2506} } @article{li_lim_puretzky_long_riehn_hallen_2018, title={DNA methylation detection using UV nano bowtie antenna enhanced Raman spectroscopy}, volume={10727}, ISSN={["1996-756X"]}, DOI={10.1117/12.2321283}, abstractNote={Methylation in DNA is a controlling factor in gene expression, embryonic development, and has been found to be important in infections and cancer. From a basic biology point of view, great heterogeneity has been found in methylation levels within tissues, so questions arises as to how and why. We show that methylated-DNA (m-DNA) can be distinguished from non-methylated (n-DNA) with nano-bowtie- and resonance- enhanced Raman spectra. By tuning the bowtie antenna to the resonance wavelength, both gains can be realized. Two additional Raman peaks in the 1200 – 1700 cm-1 band appear with methylation: one at 1239 cm-1 and the other at 1639 cm-1; a weak peak near 1000 cm-1 also appears with methylation. We also find that the two spectral features, although the latter with slight modification, can be used to distinguish the methylation state even when the DNA is denatured, as we show when we induce crystallization of the salts in the solution with increased excitation power, or allow it to happen naturally via solvent evaporation, and the DNA is trapped within the salt crystals. A comparison between liquid/solution to dried/denatured state m-DNA shows a general broadening of the larger lines and a transfer of spectral weight from the ~1470 cm-1 vibration to two higher energy lines. The applicability of the resonance-Raman in these spectra is shown by demonstrating that the Raman spectral characteristics hardly change as the Raman resonance in excitation wavelength is approached. Finally, we comment on real signal gain in this double-resonance system.}, journal={UV AND HIGHER ENERGY PHOTONICS: FROM MATERIALS TO APPLICATIONS 2018}, author={Li, Ling and Lim, Shuang Fang and Puretzky, Alexander and Long, Brandon J. N. and Riehn, Robert and Hallen, Hans}, year={2018} } @article{shojayian_irannezhad_amani_riehn_movahed_2018, title={Manipulation and control of the electrokinetic motion of a non-conductive micro-particle in microchannel by generating lateral temperature gradient}, volume={126}, url={http://dx.doi.org/10.1016/j.ijheatmasstransfer.2018.05.058}, DOI={10.1016/j.ijheatmasstransfer.2018.05.058}, abstractNote={In this article, the electrokinetic motion of a non-conductive particle immersed in an aqueous solution in a microchannel is studied; the particle is subjected to a lateral temperature gradient that is perpendicular to the direction of the applied electric field. Three-dimensional governing equations are solved numerically to simulate the motion of the particle. It is noticed that the particle undergoes lateral and rotational movements as well as a longitudinal motion. The lateral and rotational motions of the particle will be affected by a size of the particle, an applied external electric field and an initial magnitude of temperature gradient. The results show that the initial magnitude of the temperature gradient is the most significant factor. It was observed that by doubling the temperature difference, the particle travels 64 percent more in lateral direction. It also rotates 42 percent more while experiencing a doubled temperature difference. Another parameter that has been investigated is the magnitude of the electric field. By halving the magnitude of the applied electric field, the particle moves laterally 10 percent less for a constant longitudinal distance. Finally, it was discovered that the effect of size of the particle on its velocity is negligible.}, journal={International Journal of Heat and Mass Transfer}, publisher={Elsevier BV}, author={Shojayian, Mohammad and Irannezhad, Ashkan and Amani, Ehsan and Riehn, Robert and Movahed, Saeid}, year={2018}, month={Nov}, pages={861–870} } @article{roushan_azad_movahed_ray_livshits_lim_weninger_riehn_2018, title={Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement}, volume={8}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/S41598-018-28278-0}, DOI={10.1038/S41598-018-28278-0}, abstractNote={AbstractWe report that long double-stranded DNA confined to quasi-1D nanochannels undergoes superdiffusive motion under the action of the enzyme T4 DNA ligase in the presence of necessary co-factors. Inside the confined environment of the nanochannel, double-stranded DNA molecules stretch out due to self-avoiding interactions. In absence of a catalytically active enzyme, we see classical diffusion of the center of mass. However, cooperative interactions of proteins with the DNA can lead to directed motion of DNA molecules inside the nanochannel. Here we show directed motion in this configuration for three different proteins (T4 DNA ligase, MutS, E. coli DNA ligase) in the presence of their energetic co-factors (ATP, NAD+).}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Roushan, Maedeh and Azad, Zubair and Movahed, Saeid and Ray, Paul D. and Livshits, Gideon I. and Lim, Shuang Fang and Weninger, Keith R. and Riehn, Robert}, year={2018}, month={Jul} } @article{dangi_riehn_2019, title={Nanoplumbing with 2D Metamaterials}, volume={15}, ISSN={["1613-6829"]}, url={https://doi.org/10.1002/smll.201803478}, DOI={10.1002/smll.201803478}, abstractNote={AbstractComplex manipulations of DNA in a nanofluidic device require channels with branches and junctions. However, the dynamic response of DNA in such nanofluidic networks is relatively unexplored. Here, the transport of DNA in a 2D metamaterial made by arrays of nanochannel junctions is investigated. The mechanism of transport is explained as Brownian motion through an energy landscape formed by the combination of the confinement free energy of DNA and the effective potential of hydrodynamic flow, which both can be tuned independently within the device. For the quantitative understanding of DNA transport, a dynamic mean‐field model of DNA at a nanochannel junction is proposed. It is shown that the dynamics of DNA in a nanofluidic device with branched channels and junctions is well described by the model.}, number={2}, journal={SMALL}, publisher={Wiley}, author={Dangi, Saroj and Riehn, Robert}, year={2019}, month={Jan} } @article{countryman_fan_gorthi_pan_strickland_kaur_wang_lin_lei_white_et al._2018, title={Cohesin SA2 is a sequence-independent DNA-binding protein that recognizes DNA replication and repair intermediates}, volume={293}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m117.806406}, abstractNote={Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids, mediated by the cohesin protein complex, which also plays crucial roles in diverse genome maintenance pathways. Current models attribute DNA binding by cohesin to entrapment of dsDNA by the cohesin ring subunits (SMC1, SMC3, and RAD21 in humans). However, the biophysical properties and activities of the fourth core cohesin subunit SA2 (STAG2) are largely unknown. Here, using single-molecule atomic force and fluorescence microscopy imaging as well as fluorescence anisotropy measurements, we established that SA2 binds to both dsDNA and ssDNA, albeit with a higher binding affinity for ssDNA. We observed that SA2 can switch between the 1D diffusing (search) mode on dsDNA and stable binding (recognition) mode at ssDNA gaps. Although SA2 does not specifically bind to centromeric or telomeric sequences, it does recognize DNA structures often associated with DNA replication and double-strand break repair, such as a double-stranded end, single-stranded overhang, flap, fork, and ssDNA gap. SA2 loss leads to a defect in homologous recombination–mediated DNA double-strand break repair. These results suggest that SA2 functions at intermediate DNA structures during DNA transactions in genome maintenance pathways. These findings have important implications for understanding the function of cohesin in these pathways.}, number={3}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Countryman, Preston and Fan, Yanlin and Gorthi, Aparna and Pan, Hai and Strickland, Jack and Kaur, Parminder and Wang, Xuechun and Lin, Jiangguo and Lei, Xiaoying and White, Christian and et al.}, year={2018}, month={Jan}, pages={1054–1069} } @article{pan_bilinovich_kaur_riehn_wang_williams_2017, title={CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level}, volume={45}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkx548}, abstractNote={Abstract The methylcytosine-binding domain 2 (MBD2) protein recruits the nucleosome remodeling and deacetylase complex (NuRD) to methylated DNA to modify chromatin and regulate transcription. Importantly, MBD2 functions within CpG islands that contain 100s to 1000s of potential binding sites. Since NuRD physically rearranges nucleosomes, the dynamic mobility of this complex is directly related to function. In these studies, we use NMR and single-molecule atomic force microscopy and fluorescence imaging to study DNA binding dynamics of MBD2. Single-molecule fluorescence tracking on DNA tightropes containing regions with CpG-rich and CpG-free regions reveals that MBD2 carries out unbiased 1D diffusion on CpG-rich DNA but subdiffusion on CpG-free DNA. In contrast, the protein stably and statically binds to methylated CpG (mCpG) regions. The intrinsically disordered region (IDR) on MBD2 both reduces exchange between mCpG sites along the DNA as well as the dissociation from DNA, acting like an anchor that restricts the dynamic mobility of the MBD domain. Unexpectedly, MBD2 binding to methylated CpGs induces DNA bending that is augmented by the IDR region of the protein. These results suggest that MBD2 targets NuRD to unmethylated or methylated CpG islands where its distinct dynamic binding modes help maintain open or closed chromatin, respectively.}, number={15}, journal={NUCLEIC ACIDS RESEARCH}, author={Pan, Hai and Bilinovich, Stephanie M. and Kaur, Parminder and Riehn, Robert and Wang, Hong and Williams, David C., Jr.}, year={2017}, month={Sep}, pages={9164–9177} } @article{azad_riehn_2016, title={Dynamics of Large DNA Loops}, volume={110}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2015.11.3023}, DOI={10.1016/J.BPJ.2015.11.3023}, abstractNote={Dynamics of Large DNA Loops Long range interactions between genetically distant regions of DNA (>10 kbp) are not governed by the elastic energies of bending and twisting of DNA, but rather by entropic forces. Furthermore, such large scale interactions are mediated strongly by the excluded volume interactions of DNA and proteins of the cellular environment. It is not clear how these interactions locally couple to variables such as temperature, salt strength, pH, etc. in nanoconfined volumes where the monomer density of DNA is comparable to that in the cell nucleus. Using a nanofluidic device, we manipulate fluid flow to drive DNA into large loops, on the order of kilobasepairs, in a range of buffer concentrations and protein backgrounds. By analyzing the dynamics of the loop formation, steady state fluctuations, and deformation, we quantify an energy landscape. We can use this energy landscape to characterize activation barriers for proteins to form DNA loops. Once these activation barriers are overcome, we can test for enhancements in the loop elongation rate due to proteins. In addition, we can test for arrested states caused by proteins binding to specific sequences.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Azad, Zubair and Riehn, Robert}, year={2016}, month={Feb}, pages={565a} } @article{kaur_wu_lin_countryman_bradford_erie_riehn_opresko_wang_2016, title={Enhanced electrostatic force microscopy reveals higher-order DNA looping mediated by the telomeric protein TRF2}, volume={6}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/SREP20513}, DOI={10.1038/SREP20513}, abstractNote={AbstractShelterin protein TRF2 modulates telomere structures by promoting dsDNA compaction and T-loop formation. Advancement of our understanding of the mechanism underlying TRF2-mediated DNA compaction requires additional information regarding DNA paths in TRF2-DNA complexes. To uncover the location of DNA inside protein-DNA complexes, we recently developed the Dual-Resonance-frequency-Enhanced Electrostatic force Microscopy (DREEM) imaging technique. DREEM imaging shows that in contrast to chromatin with DNA wrapping around histones, large TRF2-DNA complexes (with volumes larger than TRF2 tetramers) compact DNA inside TRF2 with portions of folded DNA appearing at the edge of these complexes. Supporting coarse-grained molecular dynamics simulations uncover the structural requirement and sequential steps during TRF2-mediated DNA compaction and result in folded DNA structures with protruding DNA loops as seen in DREEM imaging. Revealing DNA paths in TRF2 complexes provides new mechanistic insights into structure-function relationships underlying telomere maintenance pathways.}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Kaur, Parminder and Wu, Dong and Lin, Jiangguo and Countryman, Preston and Bradford, Kira C. and Erie, Dorothy A. and Riehn, Robert and Opresko, Patricia L. and Wang, Hong}, year={2016}, month={Feb} } @article{lin_countryman_chen_pan_fan_jiang_kaur_miao_gurgel_you_et al._2016, title={Functional interplay between SA1 and TRF1 in telomeric DNA binding and DNA-DNA pairing}, volume={44}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkw518}, abstractNote={Proper chromosome alignment and segregation during mitosis depend on cohesion between sister chromatids. Cohesion is thought to occur through the entrapment of DNA within the tripartite ring (Smc1, Smc3 and Rad21) with enforcement from a fourth subunit (SA1/SA2). Surprisingly, cohesin rings do not play a major role in sister telomere cohesion. Instead, this role is replaced by SA1 and telomere binding proteins (TRF1 and TIN2). Neither the DNA binding property of SA1 nor this unique telomere cohesion mechanism is understood. Here, using single-molecule fluorescence imaging, we discover that SA1 displays two-state binding on DNA: searching by one-dimensional (1D) free diffusion versus recognition through subdiffusive sliding at telomeric regions. The AT-hook motif in SA1 plays dual roles in modulating non-specific DNA binding and subdiffusive dynamics over telomeric regions. TRF1 tethers SA1 within telomeric regions that SA1 transiently interacts with. SA1 and TRF1 together form longer DNA–DNA pairing tracts than with TRF1 alone, as revealed by atomic force microscopy imaging. These results suggest that at telomeres cohesion relies on the molecular interplay between TRF1 and SA1 to promote DNA–DNA pairing, while along chromosomal arms the core cohesin assembly might also depend on SA1 1D diffusion on DNA and sequence-specific DNA binding.}, number={13}, journal={NUCLEIC ACIDS RESEARCH}, author={Lin, Jiangguo and Countryman, Preston and Chen, Haijiang and Pan, Hai and Fan, Yanlin and Jiang, Yunyun and Kaur, Parminder and Miao, Wang and Gurgel, Gisele and You, Changjiang and et al.}, year={2016}, month={Jul}, pages={6363–6376} } @article{ortiz_riehn_daniels_2016, title={Nonaffine deformation under compression and decompression of a flow-stabilized solid}, volume={2016}, ISSN={["1742-5468"]}, DOI={10.1088/1742-5468/2016/08/084003}, abstractNote={Understanding the particle-scale transition from elastic deformation to plastic flow is central to making predictions about the bulk material properties and response of disordered materials. To address this issue, we perform experiments on flow-stabilized solids composed of micron-scale spheres within a microfluidic channel, in a regime where particle inertia is negligible. Each solid heap exists within a stress field imposed by the flow, and we track the positions of particles in response to single impulses of fluid-driven compression or decompression. We find that the resulting deformation field is well-decomposed into an affine field, with a constant strain profile throughout the solid, and a non-affine field. The magnitude of this non-affine response decays with the distance from the free surface in the long-time limit, suggesting that the distance from jamming plays a significant role in controlling the length scale of plastic flow. Finally, we observe that compressive pulses create more rearrangements than decompressive pulses, an effect that we quantify using the Dmin2 statistic for non-affine motion. Unexpectedly, the time scale for the compression response is shorter than for decompression at the same strain (but unequal pressure), providing insight into the coupling between deformation and cage-breaking.}, number={8}, journal={JOURNAL OF STATISTICAL MECHANICS-THEORY AND EXPERIMENT}, publisher={IOP Publishing}, author={Ortiz, Carlos P. and Riehn, Robert and Daniels, Karen E.}, year={2016}, month={Aug} } @article{zhou_riehn_2015, title={Collapse of DNA under alternating electric fields}, volume={92}, ISSN={["1550-2376"]}, DOI={10.1103/physreve.92.012714}, abstractNote={Recent studies have shown that double-stranded DNA can collapse in the presence of a strong electric field. Here we provide an in-depth study of the collapse of DNA under weak confinement in microchannels as a function of buffer strength, driving frequency, applied electric-field strength, and molecule size. We find that the critical electric field at which DNA molecules collapse (tens of kV/m) is strongly dependent on driving frequency (100-800 Hz) and molecular size (20-160 kbp), and weakly dependent on the ionic strength (8-60 mM). We argue that an apparent stretching at very high electric fields is an artifact of the finite frame time of video microscopy.}, number={1}, journal={PHYSICAL REVIEW E}, author={Zhou, Chunda and Riehn, Robert}, year={2015}, month={Jul} } @article{azad_roushan_riehn_2015, title={DNA Brushing Shoulders: Targeted Looping and Scanning of Large DNA Strands}, volume={15}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000359613700120&KeyUID=WOS:000359613700120}, DOI={10.1021/acs.nanolett.5b02476}, abstractNote={We present a nanofluidic device for targeted manipulations in the quarternary structure of single DNA molecules. We demonstrate the folding and unfolding of hairpin-shaped regions, similar to chromatin loops. These loops are stable for minutes at nanochannel junctions. We demonstrate continuous scanning of two DNA segments that occupy a common nanovolume. We present a model governing the stability of loop folds and discuss how the system achieves specific DNA configurations without operator intervention.}, number={8}, journal={Nano Letters}, author={Azad, Z. and Roushan, M. and Riehn, Robert}, year={2015}, pages={5641–5646} } @article{roushan_azad_lim_wang_riehn_2015, title={Interference of ATP with the fluorescent probes YOYO-1 and YOYO-3 modifies the mechanical properties of intercalator-stained DNA confined in nanochannels}, volume={182}, ISSN={0026-3672 1436-5073}, url={http://dx.doi.org/10.1007/S00604-015-1495-7}, DOI={10.1007/s00604-015-1495-7}, abstractNote={Intercalating fluorescent probes are widely used to visualize DNA in studies on DNA-protein interactions. Some require the presence of adenosine triphosphate (ATP). We have investigated the mechanical properties of DNA stained with the fluorescent intercalating dyes YOYO-1 and YOYO-3 as a function of ATP concentrations (up to 2 mM) by stretching single molecules in nanofluidic channels with a channel cross-section as small as roughly 100×100 nm2. The presence of ATP reduces the length of the DNA by up to 11 %. On the other hand, negligible effects are found if DNA is visualized with the minor groove-binding probe 4',6-diamidino-2-phenylindole. The apparent drop in extension under nanoconfinement is attributed to an interaction of the dye and ATP, and the resulting expulsion of YOYO-1 from the double helix.}, number={7-8}, journal={Microchimica Acta}, publisher={Springer Science and Business Media LLC}, author={Roushan, Maedeh and Azad, Zubair and Lim, Shuang Fang and Wang, Hong and Riehn, Robert}, year={2015}, month={Apr}, pages={1561–1565} } @article{ortiz_daniels_riehn_2014, title={Nonlinear elasticity of microsphere heaps}, volume={90}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000341301600006&KeyUID=WOS:000341301600006}, DOI={10.1103/physreve.90.022304}, abstractNote={Thermal fluctuations, geometric exclusion, and external driving all govern the mechanical response of dense particulate suspensions. Here, we measure the stress-strain response of quasi-two-dimensional flow-stabilized microsphere heaps in a regime in which all three effects are present using a microfluidic device. We observe that the elastic modulus and the mean interparticle separation of the heaps are tunable via the confining stress provided by the fluid flow. Furthermore, the measured stress-strain curves exhibit a universal nonlinear shape, which can be predicted from a thermal van der Waals equation of state with excluded volume. This analysis indicates that many-body interactions contribute a significant fraction of the stress supported by the heap.}, number={2}, journal={Physical Review. E, Statistical, Nonlinear, and Soft Matter Physics}, publisher={American Physical Society (APS)}, author={Ortiz, C. P. and Daniels, K. E. and Riehn, R.}, year={2014}, pages={9} } @article{roushan_kaur_karpusenko_countryman_ortiz_fang lim_wang_riehn_2014, title={Probing transient protein-mediated DNA linkages using nanoconfinement}, volume={8}, ISSN={1932-1058}, url={http://dx.doi.org/10.1063/1.4882775}, DOI={10.1063/1.4882775}, abstractNote={We present an analytic technique for probing protein-catalyzed transient DNA loops that is based on nanofluidic channels. In these nanochannels, DNA is forced in a linear configuration that makes loops appear as folds whose size can easily be quantified. Using this technique, we study the interaction between T4 DNA ligase and DNA. We find that T4 DNA ligase binding changes the physical characteristics of the DNA polymer, in particular persistence length and effective width. We find that the rate of DNA fold unrolling is significantly reduced when T4 DNA ligase and ATP are applied to bare DNA. Together with evidence of T4 DNA ligase bridging two different segments of DNA based on AFM imaging, we thus conclude that ligase can transiently stabilize folded DNA configurations by coordinating genetically distant DNA stretches.}, number={3}, journal={Biomicrofluidics}, publisher={AIP Publishing}, author={Roushan, Maedeh and Kaur, Parminder and Karpusenko, Alena and Countryman, Preston J. and Ortiz, Carlos P. and Fang Lim, Shuang and Wang, Hong and Riehn, Robert}, year={2014}, month={May}, pages={034113} } @inproceedings{lin_kaur_chen_countryman_roushan_flaherty_brennan_you_piehler_riehn_et al._2014, title={Single-molecule imaging reveals DNA-binding properties of cohesin proteins SA1 and SA2.}, volume={55}, booktitle={Environmental and Molecular Mutagenesis}, author={Lin, J. and Kaur, P. and Chen, H. and Countryman, P. and Roushan, M. and Flaherty, D. and Brennan, E. and You, C. and Piehler, J. and Riehn, R. and et al.}, year={2014}, pages={S29–29} } @article{lim_karpusenko_blumers_streng_riehn_2013, title={Chromatin modification mapping in nanochannels}, volume={7}, ISSN={1932-1058}, url={http://dx.doi.org/10.1063/1.4833257}, DOI={10.1063/1.4833257}, abstractNote={We report the simultaneous mapping of multiple histone tail modifications on chromatin that has been confined to nanofluidic channels. In these channels, chromatin is elongated, and histone modification can be detected using fluorescently tagged monoclonal antibodies. Using reconstituted chromatin with three distinct histone sources and two histone tail modification probes (H3K4me3 and H3K9ac), we were able to distinguish chromatin from the different sources. Determined ratios of the two modifications were consistent with the bulk composition of histone mixtures. We determined that the major difficulty in transitioning the mapping method to site-specific profiling within single genomic molecules is the interference of naturally aggregating, off-the shelf antibodies with the internal structure of chromatin.}, number={6}, journal={Biomicrofluidics}, publisher={AIP Publishing}, author={Lim, Shuang Fang and Karpusenko, Alena and Blumers, Ansel L. and Streng, Diana E. and Riehn, Robert}, year={2013}, month={Nov}, pages={064105} } @article{riehn_heidarpour-roushan_2013, title={DNA Looping Induced by Tubular Confinement}, volume={104}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2012.11.1425}, DOI={10.1016/j.bpj.2012.11.1425}, abstractNote={DNA looping is essential for the function and maintenance of genetic information. At short lengths the cyclization rate is limited by the semiflexible properties of DNA, while at long distances it is limited by the rapidly declining probability of colocation of the anchoring sequences of the loop. We propose that a facilitated diffusion process increases the rate of cyclization for large loops. The maze-like nuclear environment then becomes a major impediment for the required colocation. We have investigated the kinetic evolution of DNA loops (48500 bp) induced by T4 ligase inside a nanofabricated channel system with a channel cross-section of 100x100 nm2, and a few micron channel length. We found that addition of the ligase profoundly alters the behavior of DNA. In particular, ligase acts to stabilize hairpin geometries in which the extended forward and backward arms of the hairpin scan past each other. From the linear density of DNA inside the channel, we deduce that the effective excluded volume vanishes upon addition of T4 ligase and ATP. We conclude that the two strands are effectively stapled together through a large number of weak bonds involving T4 ligase. The absence of a similarly strong transformation of DNA in free solution points towards the necessity of parallel pre-alignment through the nanotube. We point to the fact that the formation of hairpins and the 1-d scanning of strands past each other solves the kinetic problem of forming a loop in a maze-like environment.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Riehn, Robert and Heidarpour-Roushan, Maedeh}, year={2013}, month={Jan}, pages={253a–254a} } @article{bret h. davis_chih-kuan tung_riehn_2013, title={SENSING DNA WITH ALTERNATING CURRENTS USING A NANOGAP SENSOR EMBEDDED IN A NANOCHANNEL DEVICE}, volume={03}, DOI={10.1142/S1793984413400072}, abstractNote={ We report an integrated nanochannel/nanoelectrode sensor for the detection of DNA using alternating currents. We find that DNA can be detected using platinum as the metal for the detecting electrodes, with a signal to noise ratio exceeding 10. We argue that the signal is at least in part electrochemical in nature, thus holds the promise to yield a sequence-dependent signal. However, we also find that for large voltages, DNA attaches irreversibly to the driving electrodes. }, number={01}, journal={Nano LIFE}, author={BRET H. DAVIS, JUNHAN PAN and CHIH-KUAN TUNG, ROBERT H. AUSTIN and RIEHN, ROBERT}, year={2013}, pages={1340007} } @article{ortiz_riehn_daniels_2013, title={Flow-driven formation of solid-like microsphere heaps}, volume={9}, ISSN={["1744-683X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84870912822&partnerID=MN8TOARS}, DOI={10.1039/c2sm26762d}, abstractNote={We observe the formation of heaps of repulsive microspheres, created by flowing a colloidal microsphere suspension towards a flat-topped ridge placed within a quasi two-dimensional microfluidic channel. This configuration allows for both shear and normal forces on the microspheres in contact with the ridge. The heaps, which are formed against the ridge, are characterized by two distinct phases: a solid-like bulk phase in the interior and a highly fluctuating, liquid-like state which exists along its leading edge. We observe that heaps only form above a critical flow velocity, vc, and that they are destroyed by thermal rearrangements when the flow ceases. We monitor the dynamics of heap formation using fluorescence video microscopy, measuring the heap volume and the angle of repose in response to microsphere deposition and erosion processes. We find that the steady state angle of repose, θf, increases as a function of inflow velocity, v∞, with a functional form .}, number={2}, journal={SOFT MATTER}, publisher={Royal Society of Chemistry (RSC)}, author={Ortiz, Carlos P. and Riehn, Robert and Daniels, Karen E.}, year={2013}, pages={543–549} } @article{karpusenko_carpenter_zhou_lim_pan_riehn_2012, title={Fluctuation modes of nanoconfined DNA}, volume={111}, ISSN={0021-8979 1089-7550}, url={http://dx.doi.org/10.1063/1.3675207}, DOI={10.1063/1.3675207}, abstractNote={We report an experimental investigation of the magnitude of length and density fluctuations in DNA that has been stretched in nanofluidic channels. We find that the experimental data can be described using a one-dimensional overdamped oscillator chain with nonzero equilibrium spring length and that a chain of discrete oscillators yields a better description than a continuous chain. We speculate that the scale of these discrete oscillators coincides with the scale at which the finite extensibility of the polymer manifests itself. We discuss how the measurement process influences the apparent measured dynamic properties, and outline requirements for the recovery of true physical quantities.}, number={2}, journal={Journal of Applied Physics}, publisher={AIP Publishing}, author={Karpusenko, Alena and Carpenter, Joshua H. and Zhou, Chunda and Lim, Shuang Fang and Pan, Junhan and Riehn, Robert}, year={2012}, month={Jan}, pages={024701} } @article{li_fang lim_puretzky_riehn_hallen_2012, title={Near-field enhanced ultraviolet resonance Raman spectroscopy using aluminum bow-tie nano-antenna}, volume={101}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84866316016&partnerID=MN8TOARS}, DOI={10.1063/1.4746747}, abstractNote={An aluminum bow-tie nano-antenna is combined with the resonance Raman effect in the deep ultraviolet to dramatically increase the sensitivity of Raman spectra to a small volume of material, such as benzene used here. We further demonstrate gradient-field Raman peaks for several strong infrared modes. We achieve a gain of ∼105 in signal intensity from the near field enhancement due to the surface plasmon resonance in the aluminum nanostructure. The on-line resonance enhancement contributes another factor of several thousands, limited by the laser line width. Thus, an overall gain of hundreds of million is achieved.}, number={11}, journal={Applied Physics Letters}, author={Li, L. and Fang Lim, S. and Puretzky, A.A. and Riehn, R. and Hallen, H.D.}, year={2012} } @article{li_lim_puretzky_riehn_hallen_2012, title={Near-field enhanced ultraviolet resonance Raman spectroscopy using aluminum bow-tie nano-antenna}, volume={101}, number={11}, journal={Applied Physics Letters}, author={Li, L. and Lim, S. F. and Puretzky, A. A. and Riehn, R. and Hallen, H. D.}, year={2012} } @article{zhou_reisner_staunton_ashan_austin_riehn_2011, title={Collapse of DNA in ac Electric Fields}, volume={106}, ISSN={["0031-9007"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-79960675475&partnerID=MN8TOARS}, DOI={10.1103/physrevlett.106.248103}, abstractNote={We report that double-stranded DNA collapses in the presence of ac electric fields at frequencies of a few hundred Hertz, and does not stretch as commonly assumed. In particular, we show that confinement-stretched DNA can collapse to about one quarter of its equilibrium length. We propose that this effect is based on finite relaxation times of the counterion cloud, and the subsequent partitioning of the molecule into mutually attractive units. We discuss alternative models of those attractive units.}, number={24}, journal={PHYSICAL REVIEW LETTERS}, author={Zhou, Chunda and Reisner, Walter W. and Staunton, Rory J. and Ashan, Amir and Austin, Robert H. and Riehn, Robert}, year={2011}, month={Jun} } @article{lim_karpusenko_sakon_hook_lamar_riehn_2011, title={DNA methylation profiling in nanochannels}, volume={5}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-80053454599&partnerID=MN8TOARS}, DOI={10.1063/1.3613671}, abstractNote={We report the profiling of the 5-methyl cytosine distribution within single genomic-sized DNA molecules at a gene-relevant resolution. This method linearizes and stretches DNA molecules by confinement to channels with a dimension of about 250×200nm2. The methylation state is detected using fluorescently labeled methyl-CpG binding domain proteins (MBD), with high signal contrast and low background. DNA barcodes consisting of methylated and non-methylated segments are generated, with both short and long concatemers demonstrating spatially resolved MBD binding. The resolution of the technique is better than 10 kbp, and single-molecule read-lengths exceeding 140 kbp have been achieved.}, number={3}, journal={Biomicrofluidics}, author={Lim, S. Fang and Karpusenko, A. and Sakon, J.J. and Hook, J.A. and Lamar, T.A. and Riehn, Robert}, year={2011} } @article{carpenter_karpusenko_pan_lim_riehn_2011, title={Density fluctuations dispersion relationship for a polymer confined to a nanotube}, volume={98}, ISSN={0003-6951 1077-3118}, url={http://dx.doi.org/10.1063/1.3602922}, DOI={10.1063/1.3602922}, abstractNote={DNA confined to rigid nanotubes shows density fluctuations around its stretched equilibrium conformation. We report an experimental investigation of the length-scale dependent dynamics of these density fluctuations. We find that for highly elongated molecules a Rouse description is consistent with observations at sufficiently large length scales. We further find that for strongly fluctuating molecules, or short length scales, such Rouse modes cannot be detected due to strong mixing of fluctuation modes.}, number={25}, journal={Applied Physics Letters}, publisher={AIP Publishing}, author={Carpenter, Joshua H. and Karpusenko, Alena and Pan, Junhan and Lim, Shuang Fang and Riehn, Robert}, year={2011}, month={Jun}, pages={253704} } @article{streng_lim_riehn_2010, title={Epigenetic Analysis of Chromatin in Nanochannels}, volume={98}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2009.12.3265}, DOI={10.1016/j.bpj.2009.12.3265}, abstractNote={Nanochannels with a diameter of about 100nm2 are a novel method for stretching DNA for genomic investigations. Such devices are implemented through standard nanolithography in fused silica. The elongation of DNA results from an interplay of steric and entropic effects. Previous applications of nanochannel stretching included sizing, restriction mapping, and observation of transcription factor binding. We show here that nanochannels can also be used to map the site-specific epigenetic state of DNA. In particular, we show here that the concept by nanoconfinement can be extended to chromatin, or DNA complexed to histones, and that the stretching is within the range expected from the de Gennes theory. We also demonstrate that the location-resolved cytidine methylation state of DNA can be mapped by specific fluorescent labeling. We will discuss the basic operation of these technique, and the application to artificial substrates with predefined epigenetic marks.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Streng, Diana E. and Lim, Shuang F. and Riehn, Robert}, year={2010}, month={Jan}, pages={600a} } @article{tung_riehn_austin_2009, title={Complementary metal oxide semiconductor compatible fabrication and characterization of parylene-C covered nanofluidic channels with integrated nanoelectrodes}, volume={3}, ISSN={["1932-1058"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77951000462&partnerID=MN8TOARS}, DOI={10.1063/1.3212074}, abstractNote={Nanochannels offer a way to align and analyze long biopolymer molecules such as DNA with high precision at potentially single basepair resolution, especially if a means to detect biomolecules in nanochannels electronically can be developed. Integration of nanochannels with electronics will require the development of nanochannel fabrication procedures that will not damage sensitive electronics previously constructed on the device. We present here a near-room-temperature fabrication technology involving parylene-C conformal deposition that is compatible with complementary metal oxide semiconductor electronic devices and present an analysis of the initial impedance measurements of conformally parylene-C coated nanochannels with integrated gold nanoelectrodes.}, number={3}, journal={BIOMICROFLUIDICS}, author={Tung, Chih-kuan and Riehn, Robert and Austin, Robert H.}, year={2009} } @article{bruinsma_riehn_2009, title={DNA Condensation by Field-Induced Non-Equilibrium Noise}, volume={10}, ISSN={["1439-7641"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-70350771174&partnerID=MN8TOARS}, DOI={10.1002/cphc.200900525}, abstractNote={AbstractDNA molecules under good solvent conditions condense when subjected to strong AC electrical fields. It is shown that AC electrical fields couple charge fluctuations to molecular shape fluctuations, which produces a non‐equilibrium noise source that can dominate over equilibrium thermal noise in the long‐wavelength limit. The field‐induced excess charge fluctuations amplify the Asakura–Oosawa fluctuation attraction force between neighboring chains, providing a mechanism for field‐induced DNA condensation.}, number={16}, journal={CHEMPHYSCHEM}, author={Bruinsma, Robijn F. and Riehn, Robert}, year={2009}, month={Nov}, pages={2871–2875} } @article{streng_lim_pan_karpusenka_riehn_2009, title={Stretching chromatin through confinement}, volume={9}, ISSN={["1473-0189"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-70349311158&partnerID=MN8TOARS}, DOI={10.1039/b909217j}, abstractNote={We present a method for the stretching of chromatin molecules in nanofluidic channels width a cross-section of about 80 x 80 nm(2), and hundreds of microns long. The stretching of chromatin to about 12 basepairs/nm enables location-resolved optical investigation of the nucleic material with a resolution of up to 6 kbp. The stretching is based on the equilibrium elongation that polymers experience when they are introduced into nanofluidic channels that are narrower than the Flory coil corresponding to the whole chromatin molecule. We investigate whether the elongation of reconstituted chromatin can be described by the de Gennes model. We compare nanofluidic stretching of bare DNA and chromatin of equal genomic length, and find that chromatin is 2.5 times more compact in its stretched state.}, number={19}, journal={LAB ON A CHIP}, author={Streng, Diana E. and Lim, Shuang Fang and Pan, Junhan and Karpusenka, Alena and Riehn, Robert}, year={2009}, pages={2772–2774} } @article{lim_riehn_tung_ryu_zhuo_dalland_austin_2009, title={Upconverting nanophosphors for bioimaging}, volume={20}, ISSN={0957-4484 1361-6528}, url={http://dx.doi.org/10.1088/0957-4484/20/40/405701}, DOI={10.1088/0957-4484/20/40/405701}, abstractNote={Upconverting nanoparticles (UCNPs) when excited in the near-infrared (NIR) region display anti-Stokes emission whereby the emitted photon is higher in energy than the excitation energy. The material system achieves that by converting two or more infrared photons into visible photons. The use of the infrared confers benefits to bioimaging because of its deeper penetrating power in biological tissues and the lack of autofluorescence. We demonstrate here sub-10 nm, upconverting rare earth oxide UCNPs synthesized by a combustion method that can be stably suspended in water when amine modified. The amine modified UCNPs show specific surface immobilization onto patterned gold surfaces. Finally, the low toxicity of the UCNPs is verified by testing on the multi-cellular C. elegans nematode.}, number={40}, journal={Nanotechnology}, publisher={IOP Publishing}, author={Lim, Shuang Fang and Riehn, Robert and Tung, Chih-kuan and Ryu, William S and Zhuo, Rui and Dalland, Joanna and Austin, Robert H}, year={2009}, month={Sep}, pages={405701} } @article{liao_galajda_riehn_ilic_puchalla_yu_craighead_austin_2008, title={Single molecule correlation spectroscopy in continuous flow mixers with zero-mode waveguides}, volume={16}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-47249143308&partnerID=MN8TOARS}, DOI={10.1364/OE.16.010077}, abstractNote={Zero-Mode Waveguides were first introduced for Fluorescence Correlation Spectroscopy at micromolar dye concentrations. We show that combining zero-mode waveguides with fluorescence correlation spectroscopy in a continuous flow mixer avoids the compression of the FCS signal due to fluid transport at channel velocities up to approximately 17 mm/s. We derive an analytic scaling relationship [equation: see text] converting this flow velocity insensitivity to improved kinetic rate certainty in time-resolved mixing experiments. Thus zero-mode waveguides make FCS suitable for direct kinetics measurements in rapid continuous flow.}, number={14}, journal={Optics Express}, author={Liao, D. and Galajda, P. and Riehn, R. and Ilic, R. and Puchalla, J.L. and Yu, H.G. and Craighead, H.G. and Austin, R.H.}, year={2008}, pages={10077–10090} } @misc{branton_deamer_marziali_bayley_benner_butler_di ventra_garaj_hibbs_huang_et al._2008, title={The potential and challenges of nanopore sequencing}, volume={26}, ISSN={["1546-1696"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-53649108801&partnerID=MN8TOARS}, DOI={10.1038/nbt.1495}, abstractNote={A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for ∼$1,000 in ∼24 h.}, number={10}, journal={NATURE BIOTECHNOLOGY}, author={Branton, Daniel and Deamer, David W. and Marziali, Andre and Bayley, Hagan and Benner, Steven A. and Butler, Thomas and Di Ventra, Massimiliano and Garaj, Slaven and Hibbs, Andrew and Huang, Xiaohua and et al.}, year={2008}, month={Oct}, pages={1146–1153} } @article{riehn_austin_sturm_2006, title={A nanofluidic railroad switch for DNA}, volume={6}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33749650219&partnerID=MN8TOARS}, DOI={10.1021/nl061137b}, abstractNote={We present a metamaterial consisting of a two-dimensional, asymmetric lattice of crossed nanochannels in fused silica, with channel diameters of 80 nm to 140 nm. When DNA is introduced, it is stretched and linearized. We show that the asymmetry in channel dimensions gives rise to a preferred direction for DNA orientation and a preferred direction for transport under dc electrophoresis. Interestingly, the preferred axis of orientation and transport can be switched by 90 degrees through application of an ac voltage. We explain the results in terms of an energy landscape for polyelectrolytes that consists of entropic and dielectrophoretic contributions and whose strength and sign can be tuned by changing the ac field strength.}, number={9}, journal={Nano Letters}, author={Riehn, R. and Austin, R.H. and Sturm, J.C.}, year={2006}, pages={1973–1976} } @article{metzler_reisner_riehn_austin_tegenfeldt_sokolov_2006, title={Diffusion mechanisms of localised knots along a polymer}, volume={76}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33751211854&partnerID=MN8TOARS}, DOI={10.1209/epl/i2006-10312-5}, abstractNote={We consider the diffusive motion of a localised knot along a linear polymer chain. In particular, we derive the mean diffusion time of the knot before it escapes from the chain once it gets close to one of the chain ends. Self-reptation of the entire chain between either end and the knot position, during which the knot is provided with free volume, leads to an L3 scaling of diffusion time; for sufficiently long chains, subdiffusion will enhance this time even more. Conversely, we propose local “breathing”, i.e., local conformational rearrangement inside the knot region (KR) and its immediate neighbourhood, as additional mechanism. The contribution of KR-breathing to the diffusion time scales only quadratically, ∼ L2, speeding up the knot escape considerably and guaranteeing finite knot mobility even for very long chains.}, number={4}, journal={Europhysics Letters}, author={Metzler, R. and Reisner, W. and Riehn, R. and Austin, R. and Tegenfeldt, J.O. and Sokolov, I.M.}, year={2006}, pages={696–702} } @article{riehn_stevenson_richards_kang_blamire_downes_cacialli_2006, title={Local probing of photocurrent and photoluminescence in a phase-separated conjugated-polymer blend by means of near-field excitation}, volume={16}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33644925553&partnerID=MN8TOARS}, DOI={10.1002/adfm.200400528}, abstractNote={AbstractIn this paper scanning near‐field microscopy is used to characterize polymer blends for photovoltaic applications, and fluorescence imaging and photoconductivity are combined to elucidate the spatial distribution and relative efficiency of current generation and photoluminescence in different domains of compositionally heterogeneous films. Focus is placed on a binary system consisting of poly[(9,9‐dioctylfluorene)‐alt‐benzothiadiazole] (F8BT) and poly[(9,9‐dioctylfluorene)‐alt‐(bis(N,N′‐(4‐butylphenyl))‐bis(N,N′‐phenyl‐1,4‐phenylenediamine))] (PFB), spun from xylene solutions, so as to obtain phase separation on micrometer and nanometer length scales. Protruding regions with diameters of about 5 μm in the topography image coincide with regions of high photocurrent (PC) and luminescence; these regions are identified as being F8BT‐rich. A general method to estimate the photoluminescence efficiency in the different domains of phase‐separated blends is proposed. As expected, lack of enhancement of the PC signal at the boundaries between protruding and lower‐lying phases indicate that these microscale boundaries play a small role in the charge generation by exciton splitting. This is consistent with the domains compositional inhomogeneity, and thus with finer phase separation within the domains. We also provide an analysis of the extent to which the metallized probe perturbs the near‐field photocurrent signal by integrating Poisson's equation. Finally, by using a Bethe–Bouwkamp model, the energy absorbed by the polymer film in the different regions is estimated.}, number={4}, journal={Advanced Functional Materials}, author={Riehn, R. and Stevenson, R. and Richards, D. and Kang, D.-J. and Blamire, M. and Downes, A. and Cacialli, F.}, year={2006}, pages={469–476} } @article{inglis_riehn_sturm_austin_2006, title={Microfluidic high gradient magnetic cell separation}, volume={99}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33646742142&partnerID=MN8TOARS}, DOI={10.1063/1.2165782}, abstractNote={Separation of blood cells by native susceptibility and by the selective attachment of magnetic beads has recently been demonstrated on microfluidic devices. We discuss the basic principles of how forces are generated via the magnetic susceptibility of an object and how microfluidics can be combined with micron-scale magnetic field gradients to greatly enhance in principle the fractionating power of magnetic fields. We discuss our efforts and those of others to build practical microfluidic devices for the magnetic separation of blood cells. We also discuss our attempts to integrate magnetic separation with other microfluidic features for developing handheld medical diagnostic tools.}, number={8}, journal={Journal of Applied Physics}, author={Inglis, D.W. and Riehn, R. and Sturm, J.C. and Austin, R.H.}, year={2006} } @article{lim_riehn_tung_tank_ryu_yao_austin_2006, title={Use of sub-10 nm diameter upconversion nanophosphors as bio-labels}, volume={950}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-40949090219&partnerID=MN8TOARS}, journal={Materials Research Society Symposium Proceedings}, author={Lim, S.F. and Riehn, R. and Tung, C.-K. and Tank, D. and Ryu, W.S. and Yao, N. and Austin, R.H.}, year={2006}, pages={174–179} } @article{riehn_austin_2006, title={Wetting micro- and nanofluidic devices using supercritical water}, volume={78}, ISSN={["0003-2700"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33747587658&partnerID=MN8TOARS}, DOI={10.1021/ac0604989}, abstractNote={We describe a method for wetting micro- and nanofluidic devices with water or any other pure liquid. The process is performed by enclosing the fluidic device in a liquid-filled cell, heating the cell to a temperature above the critical point of the liquid, and subsequent cooling of the cell to room temperature. Because the process liquid is essentially a gas during wetting, arbitrary shapes can be wetted. We demonstrate wetting of micro- and nanostructures in a fused-silica device with only a single inlet. The process is low-cost, fast, safe, and very reliable.}, number={16}, journal={ANALYTICAL CHEMISTRY}, author={Riehn, Robert and Austin, Robert H.}, year={2006}, month={Aug}, pages={5933–5934} } @article{riehn_cacialli_2005, title={A two-dimensional photonic structure made from a conjugated, fluorescent polymer}, volume={7}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-24344501327&partnerID=MN8TOARS}, DOI={10.1088/1464-4258/7/2/027}, abstractNote={We report the fabrication of a two-dimensional periodic structure made from the conjugated, fluorescent polymer poly(p-phenylene vinylene), PPV, by direct-write near-field lithography of a photosensitive precursor of the conjugated polymer. The precursor film is illuminated with ultraviolet radiation using the apertured fibre of a home-built scanning near-field optical microscope (SNOM), and unexposed areas are subsequently dissolved in methanol. The fully conjugated polymer is obtained by thermal conversion under vacuum. The structure is approximately 10 µm × 10 µm, and consists of 30 nm tall pillars on a 330 nm periodicity lattice. We present a calculation of the photonic properties and propose that structures made with the method investigated here are promising candidates for photonic crystal lasers. We also report the results of preliminary investigations aimed at increasing the pillar height.}, number={2}, journal={Journal of Optics A: Pure and Applied Optics}, author={Riehn, R. and Cacialli, F.}, year={2005} } @article{reisner_morton_riehn_wang_chou_austin_2005, title={DNA statics and dynamics in nanoscale confinement}, volume={5592}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-16644389170&partnerID=MN8TOARS}, DOI={10.1117/12.570565}, abstractNote={We present, along with theoretical scaling arguments, measurements of the equilibrium and dynamic properties of λ and T2 phage DNA molecules confined in quartz nanochannels. Such measurements serve a two-fold purpose: (1) we hope to assist in the design of future nanofluidic devices by quantifying the behavior of semiflexible polymers in confined environments and (2) we hope to test existing theories for confined semiflexible polymers.}, journal={Proceedings of SPIE - The International Society for Optical Engineering}, author={Reisner, W.W. and Morton, K. and Riehn, R. and Wang, Y.M. and Chou, S. and Austin, R.H.}, year={2005}, pages={193–200} } @article{lim_riehn_ryu_khanarian_tung_tank_austin_2006, title={In Vivo and Scanning Electron Microscopy Imaging of Upconverting Nanophosphors inCaenorhabditiselegans}, volume={6}, ISSN={1530-6984 1530-6992}, url={http://dx.doi.org/10.1021/nl0519175}, DOI={10.1021/nl0519175}, abstractNote={We show here that upconversion phosphors can be imaged both by infrared excitation and in a scanning electron microscope. We have synthesized and characterized for this work up-converting phosphor nanoparticles nonaggregated nanocrystals of size range 50-200 nm. We have investigated the optical properties of 50-200 nm nanoparticles and found a square dependence of the emitted visible fluorescence on the infrared excitation and verified that under electron excitation similar narrow band emission spectra can be obtained as is seen with IR upconversion. The viability of the nanoparticles for biological imaging was confirmed by imaging the digestive system of the nematode worm Caenorhabditis elegans, and we have confirmed using energy-dispersive X-ray analysis that the up-conversion nanoparticles can be identified in a scanning electron microscope at high spatial resolution.}, number={2}, journal={Nano Letters}, publisher={American Chemical Society (ACS)}, author={Lim, Shuang Fang and Riehn, Robert and Ryu, William S. and Khanarian, Nora and Tung, Chih-kuan and Tank, David and Austin, Robert H.}, year={2006}, month={Feb}, pages={169–174} } @article{riehn_lu_wang_lim_cox_austin_2005, title={Restriction mapping in nanofluidic devices}, volume={102}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/PNAS.0503809102}, DOI={10.1073/pnas.0503809102}, abstractNote={ We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100-200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg 2+ and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of ≈1.5 kbp in 1 min using single DNA molecules. }, number={29}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Riehn, R. and Lu, M. and Wang, Y.-M. and Lim, S. F. and Cox, E. C. and Austin, R. H.}, year={2005}, month={Jul}, pages={10012–10016} } @article{wang_tegenfeldt_reisner_riehn_guan_guo_golding_cox_sturm_austin_2005, title={Single-molecule studies of repressor-DNA interactions show long-range interactions}, volume={102}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-22244490690&partnerID=MN8TOARS}, DOI={10.1073/pnas.0502917102}, abstractNote={ We have performed single-molecule studies of GFP–LacI repressor proteins bound to bacteriophage λ DNA containing a 256 tandem lac operator insertion confined in nanochannels. An integrated photon molecular counting method was developed to determine the number of proteins bound to DNA. By using this method, we determined the saturated mean occupancy of the 256 tandem lac operators to be 13, which constitutes only 2.5% of the available sites. This low occupancy level suggests that the repressors influence each other even when they are widely separated, at distances on the order of 200 nm, or several DNA persistence lengths. }, number={28}, journal={Proceedings of the National Academy of Sciences of the United States of America}, author={Wang, Y.M. and Tegenfeldt, J.O. and Reisner, W. and Riehn, R. and Guan, X.-J. and Guo, L. and Golding, I. and Cox, E.C. and Sturm, J. and Austin, R.H.}, year={2005}, pages={9796–9801} } @article{reisner_morton_riehn_wang_yu_rosen_sturm_chou_frey_austin_2005, title={Statics and dynamics of single DNA molecules confined in nanochannels}, volume={94}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-22544464468&partnerID=MN8TOARS}, DOI={10.1103/PhysRevLett.94.196101}, abstractNote={The successful design of nanofluidic devices for the manipulation of biopolymers requires an understanding of how the predictions of soft condensed matter physics scale with device dimensions. Here we present measurements of DNA extended in nanochannels and show that below a critical width roughly twice the persistence length there is a crossover in the polymer physics.}, number={19}, journal={Physical Review Letters}, author={Reisner, W. and Morton, K.J. and Riehn, R. and Wang, Y.M. and Yu, Z. and Rosen, M. and Sturm, J.C. and Chou, S.Y. and Frey, E. and Austin, R.H.}, year={2005} } @article{inglis_riehn_austin_sturm_2004, title={Continuous microfluidic immunomagnetic cell separation}, volume={85}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-19144368234&partnerID=MN8TOARS}, DOI={10.1063/1.1823015}, abstractNote={We present a continuous-flow microfluidic device that enables cell by cell separation of cells selectively tagged with magnetic nanoparticles. The cells flow over an array of microfabricated magnetic stripes, which create a series of high magnetic field gradients that trap the magnetically labeled cells and alter their flow direction. The process was observed in real time using a low power microscope. The device has been demonstrated by the separation of leukocytes from whole human blood.}, number={21}, journal={Applied Physics Letters}, author={Inglis, D.W. and Riehn, R. and Austin, R.H. and Sturm, J.C.}, year={2004}, pages={5093–5095} } @article{cacialli_riehn_downes_latini_charas_morgado_2004, title={Fabrication of conjugated polymers nanostructures via direct near-field optical lithography}, volume={100}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-3042607949&partnerID=MN8TOARS}, DOI={10.1016/j.ultramic.2003.12.016}, abstractNote={We report our investigations into the fabrication of nanostructures of poly(p-phenylene vinylene) via direct scanning near-field lithography of its soluble precursor. Our technique is based on the spatially selective inhibition of the precursor solubility by exposure to the ultraviolet optical field present at the apex of commercially available, Au-coated near-field probes with aperture diameters between 40 and 80 nm (+/-5 nm). After development in methanol and thermal conversion under vacuum we obtain features with a minimum dimension of 160 nm. We analyse our results via tapping-mode atomic force microscopy, and find a clear phase contrast between the core and the centre of the lithographed features, corroborating the hypothesis that hard, fully insolubilised regions are surrounded by a gel-like phase, which we estimate of the order of 110-130 nm for the smallest features, by comparing our experiments with simulations carried out using a Bethe-Bouwkamp model. Use of such model also allows us to discuss the influence of probe size, tip-sample distance, and film thickness on the resolution of the lithographic process. We demonstrate the use of the technique for the direct writing of two-dimensional periodic structures with intentional defects and a periodicity relevant to applications in the visible range.}, number={3-4}, journal={Ultramicroscopy}, author={Cacialli, F. and Riehn, R. and Downes, A. and Latini, G. and Charas, A. and Morgado, J.}, year={2004}, pages={449–455} } @article{tegenfeldt_prinz_cao_chou_reisner_riehn_wang_cox_sturm_silberzan_et al._2004, title={The dynamics of genomic-length DNA molecules in 100-nm channels}, volume={101}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-4043068454&partnerID=MN8TOARS}, DOI={10.1073/pnas.0403849101}, abstractNote={We show that genomic-length DNA molecules imaged in nanochannels have an extension along the channel that scales linearly with the contour length of the polymer, in agreement with the scaling arguments developed by de Gennes for self-avoiding confined polymers. This fundamental relationship allows us to measure directly the contour length of single DNA molecules confined in the channels, and the statistical analysis of the dynamics of the polymer in the nanochannel allows us to compute the SD of the mean of the extension. This statistical analysis allows us to measure the extension of λ DNA multimers with a 130-nm SD in 1 min.}, number={30}, journal={Proceedings of the National Academy of Sciences of the United States of America}, author={Tegenfeldt, J.O. and Prinz, C. and Cao, H. and Chou, S. and Reisner, W.W. and Riehn, R. and Wang, Y.M. and Cox, E.C. and Sturm, J.C. and Silberzan, P. and et al.}, year={2004}, pages={10979–10983} } @article{riehn_charas_morgado_cacialli_2003, title={Near-field optical lithography of a conjugated polymer}, volume={82}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0037467971&partnerID=MN8TOARS}, DOI={10.1063/1.1539278}, abstractNote={We report the fabrication of poly(p-phenylene vinylene) nanostructures by direct scanning near-field lithography of its soluble precursor. The technique is based on the spatially selective inhibition of the precursor solubility by exposure to the ultraviolet optical field present at the apex of scanning near-field fiber probes with aperture diameters between 40 and 80 nm (±5 nm). After development in methanol and thermal conversion under vacuum we obtain features with a minimum dimension of 160 nm. We demonstrate the use of the technique for the direct writing of two-dimensional photonic crystals with intentional defects and a periodicity relevant to applications in the visible range. Using a Bethe–Bouwkamp model, we then discuss the influence of probe size, tip-sample distance, and film thickness on the resolution of the lithographic process. We also discuss limitations to the resolution that can arise from physical properties of the lithographic medium.}, number={4}, journal={Applied Physics Letters}, author={Riehn, R. and Charas, A. and Morgado, J. and Cacialli, F.}, year={2003}, pages={526–528} } @article{riehn_stevenson_halls_richards_kang_blamire_cacialli_2002, title={Near-field photoconductivity imaging of a conjugated polymer blend}, volume={708}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0036054160&partnerID=MN8TOARS}, journal={Materials Research Society Symposium - Proceedings}, author={Riehn, R. and Stevenson, R. and Halls, J.J.M. and Richards, D.R. and Kang, D.-J. and Blamire, M. and Cacialli, F.}, year={2002}, pages={71–76} } @article{martin_geneste_riehn_chuah_cacialli_holmes_friend_2001, title={Efficient electroluminescent poly(p-phenylene vinylene) copolymers for application in LEDs}, volume={119}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035867726&partnerID=MN8TOARS}, DOI={10.1016/S0379-6779(00)01507-1}, abstractNote={A new and efficient route for the synthesis of poly(p-phenylene vinylene)s (PPVs) containing dialkoxy substituents at the 2,3-positions of the phenylene ring of the polymer backbone has been developed. In comparison to the more classical 2,5-substitution pattern these PPV polymers show a significantly blue-shifted longest-wavelength absorption (lambda (max)) and emisson band (lambda (em)). Two statistical PPV copolymers comprising 2,3-dibutoxy and dimethyloctylsilyl- or 2,5-bis(dimethyloctylsilyl) side-chains were synthesized via the Gilch dehydrohalogenation route. Double-layer light-emitting devices (LEDs) of the configuration ITO/PEDOT/polymer/Ca were demonstrated to combine high electroluminescence efficiencies with low turn-on voltages.}, number={1-3}, journal={Synthetic Metals}, author={Martin, R.E. and Geneste, F. and Riehn, R. and Chuah, B.S. and Cacialli, F. and Holmes, A.B. and Friend, R.H.}, year={2001}, pages={43–44} } @article{stevenson_riehn_milner_richards_moons_kang_blamire_morgado_cacialli_2001, title={Erratum: Ultraviolet-visible near-field microscopy of phase-separated blends of polyfluorene-based conjugated semiconductors (Applied Physics Letters (2001) 79:833)}, volume={79}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035903428&partnerID=MN8TOARS}, DOI={10.1063/1.1404775}, abstractNote={First Page}, number={12}, journal={Applied Physics Letters}, author={Stevenson, R. and Riehn, R. and Milner, R.G. and Richards, D. and Moons, E. and Kang, D.-J. and Blamire, M. and Morgado, J. and Cacialli, F.}, year={2001} } @article{riehn_morgado_iqbal_moratti_holmes_volta_cacialli_2001, title={Fluorine-substituted poly(p-phenylenes vinylenes) copolymers}, volume={124}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035935317&partnerID=MN8TOARS}, DOI={10.1016/S0379-6779(01)00424-6}, abstractNote={Abstract We report the electrochemical, optical, and electroluminescent properties of fluorine containing poly(phenylene vinylene) random copolymers. We used 2,3,5,6-tetrafluoro-1,4-phenylene vinylene and 2-methoxy-5-(2′-ethyl-hexyloxy)-1,4-phenylene vinylene moieties, with ratios of the two copolymer units varying between 5:95 and 27:75. The electron withdrawing effect of fluorine on the conjugated π-electron system was confirmed by electrochemical measurements, which showed an increase of the oxidation and a decrease of the reduction potentials, upon increase of the fluorine content. Light emitting diodes fabricated from the copolymers showed emission in the red-orange, with lower turn-on voltages and higher electroluminescence efficiencies for lower fluorine contents. We observed maximum photoluminescence efficiencies of 6.7%.}, number={1}, journal={Synthetic Metals}, author={Riehn, R. and Morgado, J. and Iqbal, R. and Moratti, S.C. and Holmes, A.B. and Volta, S. and Cacialli, F.}, year={2001}, pages={67–69} } @article{holmes_martin_geneste_fischmeister_cacialli_riehn_friend_2001, title={New routes to monomers and polymers for LEDs}, volume={4105}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035051435&partnerID=MN8TOARS}, DOI={10.1117/12.416919}, abstractNote={The synthesis of poly(1,4-phenylene vinylene)s (PPVs) containing a 2,3-dialkoxy substitution pattern has been developed. Poly[2,3-bis(2-ethylhexyloxy)-1,4-phenylene vinylene] (BEH-PPV) 4 was prepared by Gilch polycondensation, and its optical properties were compared with the recently discussed poly(2,3-dibutoxy-1,4-phenylene vinylene) (DB-PPV) 1. The precursors for the Gilch method have traditionally been prepared by methods which have certain disadvantages. These can be overcome by the use of directed metallation reactions which are illustrated in the synthesis of some poly(2,5-disilyl-substituted 1,4-arylene vinylene) derivatives.}, journal={Proceedings of SPIE - The International Society for Optical Engineering}, author={Holmes, A.B. and Martin, R.E. and Geneste, F. and Fischmeister, C. and Cacialli, F. and Riehn, R.E. and Friend, R.H.}, year={2001}, pages={53–58} } @article{fischmeister_geneste_holmes_ma_martin_sano_tuan_cacialli_riehn_friend_2001, title={Synthesis of new building blocks for light emitting polymers}, volume={660}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035559523&partnerID=MN8TOARS}, journal={Materials Research Society Symposium - Proceedings}, author={Fischmeister, C. and Geneste, F. and Holmes, A.B. and Ma, Y. and Martin, R.E. and Sano, T. and Tuan, C.-S. and Cacialli, F. and Riehn, R.H. and Friend, R.H.}, year={2001} } @article{stevenson_riehn_milner_richards_moons_kang_blamire_morgado_cacialli_2001, title={Ultraviolet-visible near-field microscopy of phase-separated blends of polyfluorene-based conjugated semiconductors}, volume={79}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0040968740&partnerID=MN8TOARS}, DOI={10.1063/1.1389822}, abstractNote={We have used fluorescence scanning near-field microscopy to characterize polymer blends for electroluminescent applications, and thereby identify compositional nonhomogeneities. In particular, we have focused on the binary system constituted by poly(9,9′-dioctylfluorenealt-benzothiadiazole) and poly(9,9′-dioctylfluorene) (PFO), known to give efficiencies of up to 22 cd/A in light-emitting devices with suitable electrodes. Our primary aim was the assignment of the morphological features revealed in shear-force and atomic-force images of spin-coated films, and suggestive of phase separation on a 300-nm-length scale. From analysis of the fluorescence images (325 and 488 nm excitation), and quantitative correlation of optical and topographic data, we identify the raised features with PFO-rich regions. However, the limited variation in fluorescence intensity reveals a high extent of mixing within each phase on the length scale accessible in our experiment, approximately 100 nm for our focused-ion-beam-processed probe apertures.}, number={6}, journal={Applied Physics Letters}, author={Stevenson, R. and Riehn, R. and Milner, R.G. and Richards, D. and Moons, E. and Kang, D.-J. and Blamire, M. and Morgado, J. and Cacialli, F.}, year={2001}, pages={833–835} } @article{martin_geneste_chuah_fischmeister_ma_holmes_riehn_cacialli_friend_2001, title={Versatile synthesis of various conjugated aromatic homo- and copolymers}, volume={122}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0034818169&partnerID=MN8TOARS}, DOI={10.1016/S0379-6779(00)01318-7}, abstractNote={In recent years, variously substituted derivatives of poly(1,4-phenylene vinylene)s have emerged as efficient candidates for the emissive layer in polymer light emitting diodes. The synthetic routes for these polymers divide between precursor routes and those leading to fully conjugated solvent-processible polymers. The Gilch dehydrohalogenation polycondensation has largely been used for the latter class. In this presentation, we describe a novel family of 2,3-disubstituted aromatic precursors, derived from catechol, and we report their efficient polymerisation as homo- and copolymers with, for example, silyl-substituted derivatives to give materials which are highly fluorescent and serve as interesting materials in polymer LEDs.}, number={1}, journal={Synthetic Metals}, author={Martin, R.E. and Geneste, F. and Chuah, B.S. and Fischmeister, C. and Ma, Y. and Holmes, A.B. and Riehn, R. and Cacialli, F. and Friend, R.H.}, year={2001}, pages={1–5} } @article{gebel_panknin_riehn_parascandola_skorupa_2000, title={Application and improvement of the spreading resistance method for p-type 6H-SiC}, volume={338}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033723197&partnerID=MN8TOARS}, journal={Materials Science Forum}, author={Gebel, T. and Panknin, D. and Riehn, R. and Parascandola, S. and Skorupa, W.}, year={2000} } @article{martin_geneste_riehn_chuah_cacialli_friend_holmes_2000, title={Efficient blue-green light emitting poly(1,4-phenylene vinylene) copolymers}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0034696226&partnerID=MN8TOARS}, number={4}, journal={Chemical Communications}, author={Martin, R.E. and Geneste, F. and Riehn, R. and Chuah, B.S. and Cacialli, F. and Friend, R.H. and Holmes, A.B.}, year={2000}, pages={291–292} } @article{riehn_morgado_iqbal_moratti_holmes_volta_cacialli_2000, title={Electrochemical and electroluminescent properties of random copolymers of fluorine- and alkoxy-substituted poly(p-phenylene vinylene)s}, volume={33}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033749418&partnerID=MN8TOARS}, DOI={10.1021/ma992147k}, abstractNote={We report the synthesis and electroluminescent properties of random poly(phenylenevinylene) copolymers, carrying fluorine substitutions. In particular, we used 2,3,5,6-tetrafluoro-1,4-phenylenevinylene and 2-methoxy-5-(2‘-ethylhexyloxy)-1,4-phenylenevinylene moieties. The ratios of the two copolymer units were varied between 5:95 and 27:75. Electrochemical measurements showed an increase of the oxidation and a decrease of the reduction potentials of the copolymers upon increase of the fluorine content, as expected for the electron withdrawing effect of fluorine on the conjugated π-electron system. The copolymers showed emission in the red-orange region, with maximum photoluminescence efficiencies of 6.7%. We also observed lower turn-on voltages and higher electroluminescence efficiencies for lower fluorine contents.}, number={9}, journal={Macromolecules}, author={Riehn, R. and Morgado, J. and Iqbal, R. and Moratti, S.C. and Holmes, A.B. and Volta, S. and Cacialli, F.}, year={2000}, pages={3337–3341} } @article{martin_chuah_riehn_geneste_cacialli_friend_holmes_morgado_2000, title={Versatile syntheses of various homo- and copolymers of poly(1,4-arylene vinylene)s}, volume={598}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0034499504&partnerID=MN8TOARS}, journal={Materials Research Society Symposium - Proceedings}, author={Martin, R.E. and Chuah, B.S. and Riehn, R.H. and Geneste, F. and Cacialli, F. and Friend, R.H. and Holmes, A.B. and Morgado, J.}, year={2000} }