Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles

This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less supercoiling results in more movement, and more supercoiling results in less movement. In contrast to other single-molecule methodologies, the current methodology allows for studying DNA in its naturally supercoiled state with constant linking number and constant writhe. The protocol has potential for use in studying the influence of supercoils on the dynamics of DNA and its associated proteins, e.g., topoisomerase. The procedure takes ∼4 weeks.

[1]  P. Nielsen.  Sequence-selective targeting of duplex DNA by peptide nucleic acids. , 2010, Current opinion in molecular therapeutics.

[2]  Michael Griffith,et al.  SINGLE AND BIS PEPTIDE NUCLEIC ACIDS AS TRIPLEXING AGENTS : BINDING AND STOICHIOMETRY , 1995 .

[3]  S. Kowalczykowski,et al.  Exploring protein-DNA interactions in 3D using in situ construction, manipulation and visualization of individual DNA dumbbells with optical traps, microfluidics and fluorescence microscopy , 2013, Nature Protocols.

[4]  M K Cheezum,et al.  Quantitative comparison of algorithms for tracking single fluorescent particles. , 2001, Biophysical journal.

[5]  S A Stratmann,et al.  DNA replication at the single-molecule level. , 2014, Chemical Society reviews.

[6]  E. Peterman,et al.  Quantifying how DNA stretches, melts and changes twist under tension , 2011 .

[7]  Jacques Monod,et al.  The genetic control and cytoplasmic expression of “Inducibility” in the synthesis of β-galactosidase by E. coli , 1959 .

[8]  L. Oddershede,et al.  Stepwise bending of DNA by a single TATA-box binding protein. , 2006, Biophysical Journal.

[9]  David Dunlap,et al.  Direct demonstration and quantification of long-range DNA looping by the λ bacteriophage repressor , 2009, Nucleic acids research.

[10]  C. Zurla,et al.  The effect of nonspecific binding of lambda repressor on DNA looping dynamics. , 2012, Biophysical journal.

[11]  R. Landick,et al.  Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule. , 1994, Biophysical journal.

[12]  T. Strick,et al.  Twisting and stretching single DNA molecules. , 2000, Progress in biophysics and molecular biology.

[13]  Franca Esposito,et al.  Supercoiling in prokaryotic and eukaryotic DNA: changes in response to topological perturbation of plasmids in E. coli and SV40 in vitro, in nuclei and in CV-1 cells , 1987, Nucleic Acids Res..

[14]  Kim Sneppen,et al.  Single-cell Analysis of ? Immunity Regulation , 2003 .

[15]  Micah J. McCauley,et al.  Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of Actinomycin D , 2012, Nucleic acids research.

[16]  C. Bustamante,et al.  Ten years of tension: single-molecule DNA mechanics , 2003, Nature.

[17]  P. Nielsen,et al.  In vitro transcription of a torsionally constrained template. , 2002, Nucleic acids research.

[18]  D. Normanno,et al.  Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping , 2008, Nucleic acids research.

[19]  Matthew W. Pennington,et al.  Three-dimensional characterization of tethered microspheres by total internal reflection fluorescence microscopy. , 2005, Biophysical journal.

[20]  David Bensimon,et al.  Single-molecule analysis of DNA uncoiling by a type II topoisomerase , 2000, Nature.

[21]  Andrew C. Richardson,et al.  Quantifying noise in optical tweezers by allan variance. , 2009, Optics express.

[22]  Kirstine Berg-Sørensen,et al.  Optical manipulation of single molecules in the living cell. , 2014, Physical chemistry chemical physics : PCCP.

[23]  T Schlick,et al.  Internal motion of supercoiled DNA: brownian dynamics simulations of site juxtaposition. , 1998, Journal of molecular biology.

[24]  D. Dunlap,et al.  The antiparallel loops in gal DNA , 2008, Nucleic acids research.

[25]  R. Radloff,et al.  A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Kishan Dholakia,et al.  Construction and calibration of an optical trap on a fluorescence optical microscope , 2007, Nature Protocols.

[27]  P. Nielsen,et al.  Localization and dynamics of small circular DNA in live mammalian nuclei. , 2004, Nucleic acids research.

[28]  Nynke H. Dekker,et al.  Studying genomic processes at the single-molecule level: introducing the tools and applications , 2012, Nature Reviews Genetics.

[29]  Guy Nir,et al.  Dynamic analysis of a diffusing particle in a trapping potential. , 2013, Physical review. E, Statistical, nonlinear, and soft matter physics.

[30]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[31]  M. Egholm,et al.  Efficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA. , 1995, Nucleic acids research.

[32]  P. Nielsen,et al.  Structural isomers of bis-PNA bound to a target in duplex DNA. , 2001, Journal of molecular biology.

[33]  K. Sneppen,et al.  Effect of supercoiling on the λ switch , 2014, Bacteriophage.

[34]  Michelle D. Wang,et al.  Stretching DNA with optical tweezers. , 1997, Biophysical journal.

[35]  P. Nielsen,et al.  Enhanced peptide nucleic acid binding to supercoiled DNA: possible implications for DNA "breathing" dynamics. , 1996, Biochemistry.

[36]  Hernan G. Garcia,et al.  Concentration and Length Dependence of DNA Looping in Transcriptional Regulation , 2008, PLoS ONE.

[37]  W. Greenleaf,et al.  Single-molecule studies of RNA polymerase: motoring along. , 2008, Annual review of biochemistry.

[38]  M. Norgard,et al.  Increased amplification of pBR322 plasmid deoxyribonucleic acid in Escherichia coli K-12 strains RR1 and chi1776 grown in the presence of high concentrations of nucleoside , 1979, Journal of bacteriology.

[39]  D. Dunlap,et al.  Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and lambda repressors , 2022 .

[40]  J. Darlix,et al.  A single zinc finger optimizes the DNA interactions of the nucleocapsid protein of the yeast retrotransposon Ty3 , 2011, Nucleic acids research.

[41]  R. Metzler,et al.  In Vivo Facilitated Diffusion Model , 2013, PloS one.

[42]  Philip C Nelson,et al.  Tethered particle motion as a diagnostic of DNA tether length. , 2006, The journal of physical chemistry. B.

[43]  Lene B. Oddershede,et al.  Optimizing active and passive calibration of optical tweezers , 2011 .

[44]  David Bensimon,et al.  Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping , 2003, Proceedings of the National Academy of Sciences of the United States of America.