How to Think Like a Single Molecule: Obtaining Quantitative Measurements on Single DNA Molecules and Chromatin Fibers

Part of the Biological and Medical Physics, Biomedical Engineering book series (BIOMEDICAL)


There is a dichotomy between bulk, population methods and single-molecule studies to understand biological systems although both approaches generally seek to learn mechanistic information. Using single-molecule methods requires a different kind of thinking in order to achieve interpretable results. With a limited set of examples from our laboratories, we will demonstrate some successful single-molecule studies of protein–DNA interactions as well as to explain some caveats to improve future experiments. Our hope is that the reader of our chapter may learn to conceive, design, and successfully perform new single-molecule experiments by careful consideration of the desired outputs.


Estrogen Receptor Atomic Force Microscopy Optical Tweezer Chromatin Fiber Magnetic Tweezer 



We thank all our collaborators involved in the experiments described. This work was supported by GM077872 (S.H.L) and CA125514 (R.A.S.) from the National Institutes of Health.


  1. 1.
    Smith SB, Cui Y, Bustamante C (1996) Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules. Science 271:795–799CrossRefADSGoogle Scholar
  2. 2.
    Cluzel P, Lebrun A, Heller C, Lavery R, Viovy JL, Chatenay D, Caron F (1996) DNA: an extensible molecule. Science 271:792–794CrossRefADSGoogle Scholar
  3. 3.
    Williams MC, Rouzina I, Bloomfield VA (2002) Thermodynamics of DNA interactions from single molecule stretching experiments. Acc Chem Res 35:159–166CrossRefGoogle Scholar
  4. 4.
    McCauley MJ, Williams MC (2009) Optical tweezers experiments resolve distinct modes of DNA-protein binding. Biopolymers 91:265–282CrossRefGoogle Scholar
  5. 5.
    van Mameren J, Gross P, Farge G, Hooijman P, Modesti M, Falkenberg M, Wuite GJ, Peterman EJ (2009) Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging. Proc Natl Acad Sci U S A 106:18231–18236CrossRefADSGoogle Scholar
  6. 6.
    Williams MC, Rouzina I, McCauley MJ (2009) Peeling back the mystery of DNA overstretching. Proc Natl Acad Sci USA 106:18047–18048CrossRefADSGoogle Scholar
  7. 7.
    Hansma HG, Vesenka J, Siegerist C, Kelderman G, Morrett H, Sinsheimer RL, Elings V, Bustamante C, Hansma PK (1992) Reproducible imaging and dissection of plasmid DNA under liquid with the atomic force microscope. Science 256:1180–1184CrossRefADSGoogle Scholar
  8. 8.
    Allen MJ, Dong XF, O’Neill TE, Yau P, Kowalczykowski SC, Gatewood J, Balhorn R, Bradbury EM (1993) Atomic force microscope measurements of nucleosome cores assembled along defined DNA sequences. Biochemistry 32:8390–8396CrossRefGoogle Scholar
  9. 9.
    Thoma F, Koller T, Klug A (1979) Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin. J Cell Biol 83:403–427CrossRefGoogle Scholar
  10. 10.
    Leuba SH, Wheeler TB, Cheng CM, LeDuc PR, Fernandez-Sierra M, Quinones E (2009) Structure and dynamics of single DNA molecules manipulated by magnetic tweezers and or flow. Methods 47:214–222CrossRefGoogle Scholar
  11. 11.
    Zlatanova J, Leuba SH (2003) Magnetic tweezers: a sensitive tool to study DNA and chromatin at the single-molecule level. Biochem Cell Biol 81:151–159CrossRefGoogle Scholar
  12. 12.
    Pomerantz RT, Ramjit R, Gueroui Z, Place C, Anikin M, Leuba S, Zlatanova J, McAllister WT (2005) A tightly regulated molecular motor based upon T7 RNA polymerase. Nano Lett 5:1698–1703CrossRefADSGoogle Scholar
  13. 13.
    Sakata-Sogawa K, Shimamoto N (2004) RNA polymerase can track a DNA groove during promoter search. Proc Natl Acad Sci USA 101:14731–14735CrossRefADSGoogle Scholar
  14. 14.
    Harada Y, Ohara O, Takatsuki A, Itoh H, Shimamoto N, Kinosita K Jr (2001) Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase. Nature 409:113–115CrossRefADSGoogle Scholar
  15. 15.
    Kinosita K Jr, Yasuda R, Noji H, Adachi K (2000) A rotary molecular motor that can work at near 100% efficiency. Philos Trans R Soc Lond B Biol Sci 355:473–89CrossRefGoogle Scholar
  16. 16.
    Bryant Z, Stone MD, Gore J, Smith SB, Cozzarelli NR, Bustamante C (2003) Structural transitions and elasticity from torque measurements on DNA. Nature 424:338–341CrossRefADSGoogle Scholar
  17. 17.
    Gore J, Bryant Z, Stone MD, Nollmann M, Cozzarelli NR, Bustamante C (2006) Mechano chemical analysis of DNA gyrase using rotor bead tracking. Nature 439:100–104CrossRefADSGoogle Scholar
  18. 18.
    Stasiak A, Di Capua E, Koller T (1981) Elongation of duplex DNA by recA protein. J Mol Biol 151:557–564CrossRefGoogle Scholar
  19. 19.
    Bennink ML, Scharer OD, Kanaar R, Sakata-Sogawa K, Schins JM, Kanger JS, de Grooth BG, Greve J (1999) Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1. Cytometry 36:200–208CrossRefGoogle Scholar
  20. 20.
    Hegner M, Smith SB, Bustamante C (1999) Polymerization and mechanical properties of single RecA-DNA filaments. Proc Natl Acad Sci USA 96:10109–10114CrossRefADSGoogle Scholar
  21. 21.
    Leger JF, Robert J, Bourdieu L, Chatenay D, Marko JF (1998) RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations. Proc Natl Acad Sci USA 95:12295–12299CrossRefADSGoogle Scholar
  22. 22.
    Shivashankar GV, Feingold M, Krichevsky O, Libchaber A (1999) RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis. Proc Natl Acad Sci USA 96:7916–7921CrossRefADSGoogle Scholar
  23. 23.
    Anand SP, Zheng H, Bianco PR, Leuba SH, Khan SA (2007) DNA helicase activity of PcrA is not required for the displacement of RecA protein from DNA or inhibition of RecA-mediated strand exchange. J Bacteriol 189:4502–4509CrossRefGoogle Scholar
  24. 24.
    Leuba SH, Anand SP, Harp JM, Khan SA (2008) Expedient placement of two fluorescent dyes for investigating dynamic DNA protein interactions in real time. Chromosome Res 16:451–467CrossRefGoogle Scholar
  25. 25.
    Petit MA, Dervyn E, Rose M, Entian KD, McGovern S, Ehrlich SD, Bruand C (1998) PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling-circle replication. Mol Microbiol 29:261–273CrossRefGoogle Scholar
  26. 26.
    Liu S, Abbondanzieri EA, Rausch JW, Le Grice SF, Zhuang X (2008) Slide into action: dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates. Science 322:1092–1097CrossRefADSGoogle Scholar
  27. 27.
    Neuman KC, Abbondanzieri EA, Landick R, Gelles J, Block SM (2003) Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 115:437–447CrossRefGoogle Scholar
  28. 28.
    Shaevitz JW, Abbondanzieri EA, Landick R, Block SM (2003) Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 426:684–687CrossRefADSGoogle Scholar
  29. 29.
    Vassylyev DG, Artsimovitch I (2005) Tracking RNA polymerase, one step at a time. Cell 123:977–979CrossRefGoogle Scholar
  30. 30.
    Lee NK, Kapanidis AN, Koh HR, Korlann Y, Ho SO, Kim Y, Gassman N, Kim SK, Weiss S (2007) Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances. Biophys J 92:303–312CrossRefGoogle Scholar
  31. 31.
    Tomschik M, Zheng H, van Holde K, Zlatanova J, Leuba SH (2005) Fast, long-range, reversible conformational fluctuations in nucleosomes revealed by single-pair fluorescence resonance energy transfer. Proc Natl Acad Sci USA 102:3278–3283, Correction 2008 105, 10632CrossRefADSGoogle Scholar
  32. 32.
    Li G, Levitus M, Bustamante C, Widom J (2005) Rapid spontaneous accessibility of nucleosomal DNA. Nat Struct Mol Biol 12:46–53CrossRefGoogle Scholar
  33. 33.
    Li G, Widom J (2004) Nucleosomes facilitate their own invasion. Nat Struct Mol Biol 11:763–769CrossRefGoogle Scholar
  34. 34.
    Koopmans WJ, Buning R, Schmidt T, van Noort J (2009) spFRET using alternating excitation and FCS reveals progressive DNA unwrapping in nucleosomes. Biophys J 97:195–204CrossRefGoogle Scholar
  35. 35.
    Osborne CK, Bardou V, Hopp TA, Chamness GC, Hilsenbeck SG, Fuqua SA, Wong J, Allred DC, Clark GM, Schiff R (2003) Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer. J Natl Cancer Inst 95:353–361CrossRefGoogle Scholar
  36. 36.
    Okuno M, Kojima S, Matsushima-Nishiwaki R, Tsurumi H, Muto Y, Friedman SL, Moriwaki H (2004) Retinoids in cancer chemoprevention. Curr Cancer Drug Targets 4:285–298CrossRefGoogle Scholar
  37. 37.
    Jordan VC, Gapstur S, Morrow M (2001) Selective estrogen receptor modulation and reduction in risk of breast cancer, osteoporosis, and coronary heart disease. J Natl Cancer Inst 93:1449–1457CrossRefGoogle Scholar
  38. 38.
    Wang T, Xu J, Yu X, Yang R, Han ZC (2006) Peroxisome proliferator-activated receptor gamma in malignant diseases. Crit Rev Oncol Hematol 58:1–14CrossRefGoogle Scholar
  39. 39.
    Beer TM, Myrthue A (2004) Calcitriol in cancer treatment: from the lab to the clinic. Mol Cancer Ther 3:373–381Google Scholar
  40. 40.
    Cases M, Garcia-Serna R, Hettne K, Weeber M, van der Lei J, Boyer S, Mestres J (2005) Chemical and biological profiling of an annotated compound library directed to the nuclear receptor family. Curr Top Med Chem 5:763–772CrossRefGoogle Scholar
  41. 41.
    Sun S, Almaden J, Carlson TJ, Barker J, Gehring MR (2005) Assay development and data analysis of receptor-ligand binding based on scintillation proximity assay. Metab Eng 7:38–44CrossRefGoogle Scholar
  42. 42.
    Liao C, Liu B, Shi L, Zhou J, Lu XP (2005) Construction of a virtual combinatorial library using SMILES strings to discover potential structure-diverse PPAR modulators. Eur J Med Chem 40:632–640CrossRefGoogle Scholar
  43. 43.
    Schapira M, Abagyan R, Totrov M (2003) Nuclear hormone receptor targeted virtual screening. J Med Chem 46:3045–3059CrossRefGoogle Scholar
  44. 44.
    Beck V, Pfitscher A, Jungbauer A (2005) GFP-reporter for a high throughput assay to monitor estrogenic compounds. J Biochem Biophys Methods 64:19–37CrossRefGoogle Scholar
  45. 45.
    Brown PJ, Smith-Oliver TA, Charifson PS, Tomkinson NC, Fivush AM, Sternbach DD, Wade LE, Orband-Miller L, Parks DJ, Blanchard SG, Kliewer SA, Lehmann JM, Willson TM (1997) Identification of peroxisome proliferator-activated receptor ligands from a biased chemical library. Chem Biol 4:909–918CrossRefGoogle Scholar
  46. 46.
    Skretas G, Wood DW (2005) A bacterial biosensor of endocrine modulators. J Mol Biol 349:464–474CrossRefGoogle Scholar
  47. 47.
    Lin CY, Vega VB, Thomsen JS, Zhang T, Kong SL, Xie M, Chiu KP, Lipovich L, Barnett DH, Stossi F, Yeo A, George J, Kuznetsov VA, Lee YK, Charn TH, Palanisamy N, Miller LD, Cheung E, Katzenellenbogen BS, Ruan Y, Bourque G, Wei CL, Liu ET (2007) Whole-genome cartography of estrogen receptor alpha binding sites. PLoS Genet 3:e87CrossRefGoogle Scholar
  48. 48.
    Htun H, Holth LT, Walker D, Davie JR, Hager GL (1999) Direct visualization of the human estrogen receptor alpha reveals a role for ligand in the nuclear distribution of the receptor. Mol Biol Cell 10:471–486Google Scholar
  49. 49.
    King WJ, Greene GL (1984) Monoclonal antibodies localize oestrogen receptor in the nuclei of target cells. Nature 307:745–747CrossRefADSGoogle Scholar
  50. 50.
    Martinez ED, Rayasam GV, Dull AB, Walker DA, Hager GL (2005) An estrogen receptor chimera senses ligands by nuclear translocation. J Steroid Biochem Mol Biol 97:307–321CrossRefGoogle Scholar
  51. 51.
    Picard D, Kumar V, Chambon P, Yamamoto KR (1990) Signal transduction by steroid hormones: nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell Regul 1:291–299Google Scholar
  52. 52.
    Press MF, Xu SH, Wang JD, Greene GL (1989) Subcellular distribution of estrogen receptor and progesterone receptor with and without specific ligand. Am J Pathol 135:857–864Google Scholar
  53. 53.
    Stenoien DL, Mancini MG, Patel K, Allegretto EA, Smith CL, Mancini MA (2000) Subnuclear trafficking of estrogen receptor-alpha and steroid receptor coactivator-1. Mol Endocrinol 14:518–534CrossRefGoogle Scholar
  54. 54.
    Stenoien DL, Nye AC, Mancini MG, Patel K, Dutertre M, O’Malley BW, Smith CL, Belmont AS, Mancini MA (2001) Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor alpha-coactivator complexes in living cells. Mol Cell Biol 21:4404–4412CrossRefGoogle Scholar
  55. 55.
    Berno V, Amazit L, Hinojos C, Zhong J, Mancini MG, Sharp ZD, Mancini MA (2008) Activation of estrogen receptor-alpha by E2 or EGF induces temporally distinct patterns of large-scale chromatin modification and mRNA transcription. PLoS One 3:e2286CrossRefADSGoogle Scholar
  56. 56.
    Sharp ZD, Mancini MG, Hinojos CA, Dai F, Berno V, Szafran AT, Smith KP, Lele TP, Ingber DE, Mancini MA (2006) Estrogen-receptor-alpha exchange and chromatin dynamics are ligand- and domain-dependent. J Cell Sci 119:4101–4116CrossRefGoogle Scholar
  57. 57.
    Metivier R, Penot G, Carmouche RP, Hubner MR, Reid G, Denger S, Manu D, Brand H, Kos M, Benes V, Gannon F (2004) Transcriptional complexes engaged by apo-estrogen receptor-alpha isoforms have divergent outcomes. EMBO J 23:3653–3666CrossRefGoogle Scholar
  58. 58.
    Metivier R, Penot G, Hubner MR, Reid G, Brand H, Kos M, Gannon F (2003) Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter. Cell 115:751–763CrossRefGoogle Scholar
  59. 59.
    Kraus WL, Kadonaga JT (1998) p300 and estrogen receptor cooperatively activate transcription via differential enhancement of initiation and reinitiation. Genes Dev 12:331–342CrossRefGoogle Scholar
  60. 60.
    Kangaspeska S, Stride B, Metivier R, Polycarpou-Schwarz M, Ibberson D, Carmouche RP, Benes V, Gannon F, Reid G (2008) Transient cyclical methylation of promoter DNA. Nature 452:112–115CrossRefADSGoogle Scholar
  61. 61.
    Metivier R, Gallais R, Tiffoche C, Le Peron C, Jurkowska RZ, Carmouche RP, Ibberson D, Barath P, Demay F, Reid G, Benes V, Jeltsch A, Gannon F, Salbert G (2008) Cyclical DNA methylation of a transcriptionally active promoter. Nature 452:45–50CrossRefADSGoogle Scholar
  62. 62.
    Lowary PT, Widom J (1998) New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning. J Mol Biol 276:19–42CrossRefGoogle Scholar
  63. 63.
    Satchwell SC, Drew HR, Travers AA (1986) Sequence periodicities in chicken nucleosome core DNA. J Mol Biol 191:659–675CrossRefGoogle Scholar
  64. 64.
    Wang JP, Widom J (2005) Improved alignment of nucleosome DNA sequences using a mixture model. Nucleic Acids Res 33:6743–6755CrossRefGoogle Scholar
  65. 65.
    Rich RL, Hoth LR, Geoghegan KF, Brown TA, LeMotte PK, Simons SP, Hensley P, Myszka DG (2002) Kinetic analysis of estrogen receptor/ligand interactions. Proc Natl Acad Sci USA 99:8562–8567CrossRefADSGoogle Scholar
  66. 66.
    Rasnik I, McKinney SA, Ha T (2006) Nonblinking and long-lasting single-molecule fluorescence imaging. Nat Methods 3:891–893CrossRefGoogle Scholar
  67. 67.
    Aitken CE, Marshall RA, Puglisi JD (2008) An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 94:1826–1835CrossRefGoogle Scholar
  68. 68.
    Hohng S, Joo C, Ha T (2004) Single-molecule three-color FRET. Biophys J 87:1328–1337CrossRefGoogle Scholar
  69. 69.
    Kapanidis AN, Lee NK, Laurence TA, Doose S, Margeat E, Weiss S (2004) Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc Natl Acad Sci USA 101:8936–8941CrossRefADSGoogle Scholar
  70. 70.
    Leuba SH, Yang G, Robert C, Samori B, van Holde K, Zlatanova J, Bustamante C (1994) Three-dimensional structure of extended chromatin fibers as revealed by tapping-mode scanning force microscopy. Proc Natl Acad Sci USA 91:11621–11625CrossRefADSGoogle Scholar

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© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Departments of Cell Biology and Physiology and Bioengineering, Hillman Cancer Center, Petersen Institute of NanoScience and Engineering and University of Pittsburgh Cancer InstituteUniversity of Pittsburgh School of Medicine and Swanson School of EngineeringPittsburghUSA

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