Studying genomic processes at the single-molecule level: introducing the tools and applications

Key Points

  • We present key examples of the use of single-molecule approaches to study transcription, translation, splicing and replication.

  • We highlight the particular advantages of using single-molecule approaches for the study of genome processing.

  • We provide an overview of the force manipulation and fluorescent techniques used to study genomic processes.

  • We highlight how single-molecule studies of transcription have provided novel insights into initiation, elongation and termination.

  • We discuss how in the field of translation, single-molecule methods have been used to dissect aspects of initiation, elongation, termination and protein folding.

  • Novel single-molecule fluorescence assays have allowed studies of splicing and nuclear export in unprecedented detail.

  • We review how single-molecule techniques provided surprising insights on the stoichiometry and dynamics of the replisome.

  • We conclude with an overview of challenges and future directions in the application of approaches to genomic processes.

Abstract

To understand genomic processes such as transcription, translation or splicing, we need to be able to study their spatial and temporal organization at the molecular level. Single-molecule approaches provide this opportunity, allowing researchers to monitor molecular conformations, interactions or diffusion quantitatively and in real time in purified systems and in the context of the living cell. This Review introduces the types of application of single-molecule approaches that can enhance our understanding of genome function.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Studies of RNA polymerase at the single-molecule level.
Figure 2: Studies of the ribosome and translation at the single-molecule level.
Figure 3: Observations of nuclear export and splicing at the single-molecule level.
Figure 4: Replisome architecture and dynamics.
Figure 5: Genome processing taking place inside a cell nucleus and potential ways of monitoring it at the single-molecule level.

References

  1. 1

    Pareek, C. S., Smoczynski, R. & Tretyn, A. Sequencing technologies and genome sequencing. J. Appl. Genet. 52, 413–435 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2

    Forget, A. L. & Kowalczykowski, S. C. Single-molecule imaging brings Rad51 nucleoprotein filaments into focus. Trends Cell Biol. 20, 269–276 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3

    Finkelstein, I. J. & Greene, E. C. Single molecule studies of homologous recombination. Mol. Biosyst. 4, 1094–1104 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4

    Vinograd, J., Lebowitz, J., Radloff, R., Watson, R. & Laipis, P. The twisted circular form of polyoma viral DNA. Proc. Natl Acad. Sci. USA 53, 1104–1111 (1965).

    CAS  Article  Google Scholar 

  5. 5

    Sebring, E. D., Kelly, T. J. Jr, Thoren, M. M. & Salzman, N. P. Structure of replicating simian virus 40 deoxyribonucleic acid molecules. J. Virol. 8, 478–490 (1971).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Ostrander, E. A., Benedetti, P. & Wang, J. C. Template supercoiling by a chimera of yeast GAL4 protein and phage T7 RNA polymerase. Science 249, 1261–1265 (1990).

    CAS  Article  Google Scholar 

  7. 7

    Neher, E. & Sakmann, B. Patch clamp techniques for studying ionic channels in excitable membranes. Annu. Rev. Physiol. 46, 455–472 (1984).

    Article  Google Scholar 

  8. 8

    Orrit, M. & Bernard, J. Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal. Phys. Rev. Lett. 65, 2716–2719 (1990).

    CAS  Article  Google Scholar 

  9. 9

    Betzig, E. & Chichester, R. J. Single molecules observed by near-field scanning optical microscopy. Science 262, 1422–1425 (1993).

    CAS  Article  Google Scholar 

  10. 10

    Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11

    Wang, M. D. et al. Force and velocity measured for single molecules of RNA polymerase. Science 282, 902–907 (1998).

    CAS  Article  Google Scholar 

  12. 12

    Dutta, D., Shatalin, K., Epshtein, V., Gottesman, M. E. & Nudler, E. Linking RNA polymerase backtracking to genome instability in E. coli. Cell 146, 533–543 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13

    Koster, D. A., Croquette, V., Dekker, C., Shuman, S. & Dekker, N. H. Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB. Nature 434, 671–674 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Bormuth, V., Varga, V., Howard, J. & Schaffer, E. Protein friction limits diffusive and directed movements of kinesin motors on microtubules. Science 325, 870–873 (2009).

    CAS  Article  Google Scholar 

  15. 15

    Schafer, D. A., Gelles, J., Sheetz, M. P. & Landick, R. Transcription by single molecules of RNA polymerase observed by light microscopy. Nature 352, 444–448 (1991).

    CAS  Article  Google Scholar 

  16. 16

    Friedman, L. J. & Gelles, J. Mechanism of transcription initiation at an activator-dependent promoter defined by single-molecule observation. Cell 148, 679–689 (2012). This paper describes studies using an in vitro TIRF microscopy assay of the association and dissociation of the bacterial RNA Pol σ54 factor during the transition from initiation to elongation.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    Kapanidis, A. N. et al. Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science 314, 1144–1147 (2006). This was the first in vitro experimental complete study showing the scrunching mechanism of RNA Pol initiation by smFRET.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Revyakin, A., Liu, C., Ebright, R. H. & Strick, T. R. Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science 314, 1139–1143 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Treutlein, B. et al. Dynamic architecture of a minimal RNA polymerase II open promoter complex. Mol. Cell 46, 136–146 (2012).

    CAS  Article  Google Scholar 

  20. 20

    Abbondanzieri, E. A., Greenleaf, W. J., Shaevitz, J. W., Landick, R. & Block, S. M. Direct observation of base-pair stepping by RNA polymerase. Nature 438, 460–465 (2005). This paper reports the first force spectroscopy study of RNA Pol at the base-pair resolution, using optical tweezers.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Neuman, K. C., Abbondanzieri, E. A., Landick, R., Gelles, J. & Block, S. M. Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking. Cell 115, 437–447 (2003).

    CAS  Article  Google Scholar 

  22. 22

    Herbert, K. M. et al. Sequence-resolved detection of pausing by single RNA polymerase molecules. Cell 125, 1083–1094 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Shaevitz, J. W., Abbondanzieri, E. A., Landick, R. & Block, S. M. Backtracking by single RNA polymerase molecules observed at near-base-pair resolution. Nature 426, 684–687 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Galburt, E. A. et al. Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner. Nature 446, 820–823 (2007).

    CAS  Article  PubMed  Google Scholar 

  25. 25

    Depken, M., Galburt, E. A. & Grill, S. W. The origin of short transcriptional pauses. Biophys. J. 96, 2189–2193 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26

    Mejia, Y. X., Mao, H., Forde, N. R. & Bustamante, C. Thermal probing of E. coli RNA polymerase off-pathway mechanisms. J. Mol. Biol. 382, 628–637 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27

    Zhou, J., Ha, K. S., La Porta, A., Landick, R. & Block, S. M. Applied force provides insight into transcriptional pausing and its modulation by transcription factor NusA. Mol. Cell 44, 635–646 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Herbert, K. M. et al. E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase. J. Mol. Biol. 399, 17–30 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    Dalal, R. V. et al. Pulling on the nascent RNA during transcription does not alter kinetics of elongation or ubiquitous pausing. Mol. Cell 23, 231–239 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30

    Maoileidigh, D. O., Tadigotla, V. R., Nudler, E. & Ruckenstein, A. E. A unified model of transcription elongation: what have we learned from single-molecule experiments? Biophys. J. 100, 1157–1166 (2011).

    CAS  Article  Google Scholar 

  31. 31

    Voliotis, M., Cohen, N., Molina-Paris, C. & Liverpool, T. B. Fluctuations, pauses, and backtracking in DNA transcription. Biophys. J. 94, 334–348 (2008).

    CAS  Article  Google Scholar 

  32. 32

    Hodges, C., Bintu, L., Lubkowska, L., Kashlev, M. & Bustamante, C. Nucleosomal fluctuations govern the transcription dynamics of RNA polymerase II. Science 325, 626–628 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33

    Darzacq, X. et al. In vivo dynamics of RNA polymerase II transcription. Nature Struct. Mol. Biol. 14, 796–806 (2007).

    CAS  Article  Google Scholar 

  34. 34

    Larson, M. H., Greenleaf, W. J., Landick, R. & Block, S. M. Applied force reveals mechanistic and energetic details of transcription termination. Cell 132, 971–982 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. 35

    Gusarov, I. & Nudler, E. The mechanism of intrinsic transcription termination. Mol. Cell 3, 495–504 (1999).

    CAS  Article  Google Scholar 

  36. 36

    Petrov, A. et al. Dynamics of the translational machinery. Curr. Opin. Struct. Biol. 21, 137–145 (2011).

    CAS  Article  Google Scholar 

  37. 37

    Marshall, R. A., Aitken, C. E. & Puglisi, J. D. GTP hydrolysis by IF2 guides progression of the ribosome into elongation. Mol. Cell 35, 37–47 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38

    Cornish, P. V., Ermolenko, D. N., Noller, H. F. & Ha, T. Spontaneous intersubunit rotation in single ribosomes. Mol. Cell 30, 578–588 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39

    Aitken, C. E. & Puglisi, J. D. Following the intersubunit conformation of the ribosome during translation in real time. Nature Struct. Mol. Biol. 17, 793–800 (2010). This study used smFRET to follow the inter-subunit conformation of the ribosome during translation in real time.

    CAS  Article  Google Scholar 

  40. 40

    Munro, J. B., Altman, R. B., O'Connor, N. & Blanchard, S. C. Identification of two distinct hybrid state intermediates on the ribosome. Mol. Cell 25, 505–517 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. 41

    Wen, J. D. et al. Following translation by single ribosomes one codon at a time. Nature 452, 598–603 (2008). This was the first in vitro observation of translating ribosomes using single-molecule force spectroscopy.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42

    Namy, O., Moran, S. J., Stuart, D. I., Gilbert, R. J. & Brierley, I. A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting. Nature 441, 244–247 (2006).

    CAS  Article  Google Scholar 

  43. 43

    Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. Science 323, 133–138 (2009).

    CAS  Article  Google Scholar 

  44. 44

    Uemura, S. et al. Real-time tRNA transit on single translating ribosomes at codon resolution. Nature 464, 1012–1017 (2010). This paper provides a demonstration of the use of ZMW to study in vitro translation in the presence of a high concentration of labelled tRNAs.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45

    Gao, Y. G. et al. The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 326, 694–699 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46

    Sternberg, S. H., Fei, J., Prywes, N., McGrath, K. A. & Gonzalez, R. L. Jr. Translation factors direct intrinsic ribosome dynamics during translation termination and ribosome recycling. Nature Struct. Mol. Biol. 16, 861–868 (2009).

    CAS  Article  Google Scholar 

  47. 47

    Kaiser, C. M., Goldman, D. H., Chodera, J. D., Tinoco, I. Jr & Bustamante, C. The ribosome modulates nascent protein folding. Science 334, 1723–1727 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. 48

    Yang, W., Gelles, J. & Musser, S. M. Imaging of single-molecule translocation through nuclear pore complexes. Proc. Natl Acad. Sci. USA 101, 12887–12892 (2004).

    CAS  Article  Google Scholar 

  49. 49

    Kubitscheck, U. et al. Nuclear transport of single molecules: dwell times at the nuclear pore complex. J. Cell Biol. 168, 233–243 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Lowe, A. R. et al. Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking. Nature 467, 600–603 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. 51

    Milles, S. & Lemke, E. A. Single molecule study of the intrinsically disordered FG-repeat nucleoporin 153. Biophys. J. 101, 1710–1719 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. 52

    Kowalczyk, S. W. et al. Single-molecule transport across an individual biomimetic nuclear pore complex. Nature Nanotechnol. 6, 433–438 (2011).

    CAS  Article  Google Scholar 

  53. 53

    Grunwald, D. & Singer, R. H. In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport. Nature 467, 604–607 (2010). This is a demonstration of tracking mRNA export through the nuclear pore in live cells using a super-registration approach.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Abelson, J. et al. Conformational dynamics of single pre-mRNA molecules during in vitro splicing. Nature Struct. Mol. Biol. 17, 504–512 (2010).

    CAS  Article  Google Scholar 

  55. 55

    Karunatilaka, K. S., Solem, A., Pyle, A. M. & Rueda, D. Single-molecule analysis of Mss116-mediated group II intron folding. Nature 467, 935–939 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. 56

    Hoskins, A. A. et al. Ordered and dynamic assembly of single spliceosomes. Science 331, 1289–1295 (2011). This is a comprehensive study of spliceosome assembly using cell extract and multicolour TIRF microscopy.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. 57

    Waks, Z., Klein, A. M. & Silver, P. A. Cell-to-cell variability of alternative RNA splicing. Mol. Syst. Biol. 7, 506 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58

    Vargas, D. Y. et al. Single-molecule imaging of transcriptionally coupled and uncoupled splicing. Cell 147, 1054–1065 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. 59

    O'Donnell, M. Replisome architecture and dynamics in Escherichia coli. J. Biol. Chem. 281, 10653–10656 (2006).

    CAS  Article  Google Scholar 

  60. 60

    Bates, D. The bacterial replisome: back on track? Mol. Microbiol. 69, 1341–1348 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  61. 61

    Yao, N. Y. & O'Donnell, M. SnapShot: the replisome. Cell 141, 1088–1088.e1 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  62. 62

    Benkovic, S. J., Valentine, A. M. & Salinas, F. Replisome-mediated DNA replication. Annu. Rev. Biochem. 70, 181–208 (2001).

    CAS  Article  PubMed  Google Scholar 

  63. 63

    Johnson, A. & O'Donnell, M. Cellular DNA replicases: components and dynamics at the replication fork. Annu. Rev. Biochem. 74, 283–315 (2005).

    CAS  Article  Google Scholar 

  64. 64

    Maier, B., Bensimon, D. & Croquette, V. Replication by a single DNA polymerase of a stretched single-stranded DNA. Proc. Natl Acad. Sci. USA 97, 12002–12007 (2000).

    CAS  Article  Google Scholar 

  65. 65

    Manosas, M., Spiering, M. M., Zhuang, Z., Benkovic, S. J. & Croquette, V. Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome. Nature Chem. Biol. 5, 904–912 (2009). This is a thorough investigation of the T4 primosome activity on the single-molecule level.

    CAS  Article  Google Scholar 

  66. 66

    Ribeck, N., Kaplan, D. L., Bruck, I. & Saleh, O. A. DNAB helicase activity is modulated by DNA geometry and force. Biophys. J. 99, 2170–2179 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  67. 67

    Johnson, D. S., Bai, L., Smith, B. Y., Patel, S. S. & Wang, M. D. Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell 129, 1299–1309 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  68. 68

    Zhou, R. et al. SSB functions as a sliding platform that migrates on DNA via reptation. Cell 146, 222–232 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  69. 69

    Manosas, M., Spiering, M. M., Ding, F., Croquette, V. & Benkovic, S. J. Collaborative coupling between polymerase and helicase for leading-strand synthesis. Nucleic Acids Res. 40, 6187–6198 (2012).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  70. 70

    Lee, J. B. et al. DNA primase acts as a molecular brake in DNA replication. Nature 439, 621–624 (2006).

    CAS  Article  Google Scholar 

  71. 71

    Tanner, N. A. et al. Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nature Struct. Mol. Biol. 15, 170–176 (2008).

    CAS  Article  Google Scholar 

  72. 72

    Yardimci, H., Loveland, A. B., Habuchi, S., van Oijen, A. M. & Walter, J. C. Uncoupling of sister replisomes during eukaryotic DNA replication. Mol. Cell 40, 834–840 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  73. 73

    Pandey, M. et al. Coordinating DNA replication by means of priming loop and differential synthesis rate. Nature 462, 940–943 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  74. 74

    McInerney, P., Johnson, A., Katz, F. & O'Donnell, M. Characterization of a triple DNA polymerase replisome. Mol. Cell 27, 527–538 (2007).

    CAS  Article  Google Scholar 

  75. 75

    Lovett, S. T. Polymerase switching in DNA replication. Mol. Cell 27, 523–526 (2007).

    CAS  Article  Google Scholar 

  76. 76

    Xie, X. S., Choi, P. J., Li, G. W., Lee, N. K. & Lia, G. Single-molecule approach to molecular biology in living bacterial cells. Annu. Rev. Biophys. 37, 417–444 (2008).

    CAS  Article  Google Scholar 

  77. 77

    Reyes-Lamothe, R., Sherratt, D. J. & Leake, M. C. Stoichiometry and architecture of active DNA replication machinery in Escherichia coli. Science 328, 498–501 (2010). This was the first paper that determined the stoichiometry of the replisome in the living cell using single-molecule techniques.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  78. 78

    Lia, G., Michel, B. & Allemand, J. F. Polymerase exchange during Okazaki fragment synthesis observed in living cells. Science 335, 328–331 (2012).

    CAS  Article  Google Scholar 

  79. 79

    Georgescu, R. E., Kurth, I. & O'Donnell, M. E. Single-molecule studies reveal the function of a third polymerase in the replisome. Nature Struct. Mol. Biol. 19, 113–116 (2012).

    CAS  Article  Google Scholar 

  80. 80

    McHenry, C. S. D. N. A. Replicases from a bacterial perspective. Annu. Rev. Biochem. 80, 403–436 (2011).

    CAS  Article  Google Scholar 

  81. 81

    Koster, D. A., Crut, A., Shuman, S., Bjornsti, M. A. & Dekker, N. H. Cellular strategies for regulating DNA supercoiling: a single-molecule perspective. Cell 142, 519–530 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  82. 82

    Lipfert, J., Kerssemakers, J. W., Jager, T. & Dekker, N. H. Magnetic torque tweezers: measuring torsional stiffness in DNA and RecA-DNA filaments. Nature Methods 7, 977–980 (2010).

    CAS  Article  Google Scholar 

  83. 83

    Lipfert, J., Wiggin, M., Kerssemakers, J. W., Pedaci, F. & Dekker, N. H. Freely orbiting magnetic tweezers to directly monitor changes in the twist of nucleic acids. Nature Commun. 2, 439 (2011).

    Article  CAS  Google Scholar 

  84. 84

    Gore, J. et al. Mechanochemical analysis of DNA gyrase using rotor bead tracking. Nature 439, 100–104 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  85. 85

    Bryant, Z. et al. Structural transitions and elasticity from torque measurements on DNA. Nature 424, 338–341 (2003).

    CAS  Article  Google Scholar 

  86. 86

    La Porta, A. & Wang, M. D. Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles. Phys. Rev. Lett. 92, 190801 (2004).

    Article  CAS  Google Scholar 

  87. 87

    Comstock, M. J., Ha, T. & Chemla, Y. R. Ultrahigh-resolution optical trap with single-fluorophore sensitivity. Nature Methods 8, 335–340 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  88. 88

    Hohng, S. et al. Fluorescence-force spectroscopy maps two-dimensional reaction landscape of the Holliday junction. Science 318, 279–283 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  89. 89

    Liu, R., Garcia-Manyes, S., Sarkar, A., Badilla, C. L. & Fernandez, J. M. Mechanical characterization of protein l in the low-force regime by electromagnetic tweezers/evanescent nanometry. Biophys. J. 96, 3810–3821 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  90. 90

    Yan, J. et al. Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics. Mol. Biol. Cell 18, 464–474 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  91. 91

    Yeom, K. H. et al. Single-molecule approach to immunoprecipitated protein complexes: insights into miRNA uridylation. EMBO Rep. 12, 690–696 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  92. 92

    Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  93. 93

    Ribeck, N. & Saleh, O. A. Multiplexed single-molecule measurements with magnetic tweezers. Rev. Sci. Instrum. 79, 094301 (2008).

    Article  CAS  Google Scholar 

  94. 94

    Umbarger, M. A. et al. The three-dimensional architecture of a bacterial genome and its alteration by genetic perturbation. Mol. Cell 44, 252–264 (2011).

    CAS  Article  Google Scholar 

  95. 95

    Schoen, I., Ries, J., Klotzsch, E., Ewers, H. & Vogel, V. Binding-activated localization microscopy of DNA structures. Nano Lett. 11, 4008–4011 (2011).

    CAS  Article  Google Scholar 

  96. 96

    Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods 5, 877–879 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  97. 97

    Trcek, T. et al. Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nature Protoc. 7, 408–419 (2012).

    CAS  Article  Google Scholar 

  98. 98

    Paige, J. S., Wu, K. Y. & Jaffrey, S. R. RNA mimics of green fluorescent protein. Science 333, 642–646 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  99. 99

    Lee, S. F., Thompson, M. A., Schwartz, M. A., Shapiro, L. & Moerner, W. E. Super-resolution imaging of the nucleoid-associated protein HU in Caulobacter crescentus. Biophys. J. 100, L31–L33 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  100. 100

    Wang, W., Li, G. W., Chen, C., Xie, X. S. & Zhuang, X. Chromosome organization by a nucleoid-associated protein in live bacteria. Science 333, 1445–1449 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  101. 101

    Fernandez-Suarez, M. & Ting, A. Y. Fluorescent probes for super-resolution imaging in living cells. Nature Rev. Mol. Cell Biol. 9, 929–943 (2008).

    CAS  Article  Google Scholar 

  102. 102

    Eyckmans, J., Boudou, T., Yu, X. & Chen, C. S. A hitchhiker's guide to mechanobiology. Dev. Cell 21, 35–47 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  103. 103

    Muller, D. J. & Dufrene, Y. F. Force nanoscopy of living cells. Curr. Biol. 21, R212–R216 (2011).

    Article  CAS  Google Scholar 

  104. 104

    Legant, W. R. et al. Microfabricated tissue gauges to measure and manipulate forces from 3D microtissues. Proc. Natl Acad. Sci. USA 106, 10097–10102 (2009).

    CAS  Article  Google Scholar 

  105. 105

    Xie, C., Hanson, L., Cui, Y. & Cui, B. Vertical nanopillars for highly localized fluorescence imaging. Proc. Natl Acad. Sci. USA 108, 3894–3899 (2011).

    CAS  Article  Google Scholar 

  106. 106

    Zhang, J. Microsystems for cellular force measurement: a review. J. Micromech. Microeng. 21, 054003 (2011).

    Article  Google Scholar 

  107. 107

    Dufrene, Y. F. et al. Five challenges to bringing single-molecule force spectroscopy into living cells. Nature Methods 8, 123–127 (2011).

    CAS  Article  Google Scholar 

  108. 108

    Wang, Y., Meng, F. & Sachs, F. Genetically encoded force sensors for measuring mechanical forces in proteins. Commun. Integr. Biol. 4, 385–390 (2011).

    CAS  Google Scholar 

  109. 109

    Sims, P. A. & Xie, X. S. Probing dynein and kinesin stepping with mechanical manipulation in a living cell. ChemPhysChem 10, 1511–1516 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  110. 110

    Celedon, A., Hale, C. M. & Wirtz, D. Magnetic manipulation of nanorods in the nucleus of living cells. Biophys. J. 101, 1880–1886 (2011).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  111. 111

    Hu, S. et al. Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells. Am. J. Physiol. Cell Physiol. 285, C1082–C1090 (2003).

    CAS  Article  Google Scholar 

  112. 112

    Grashoff, C. et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466, 263–266 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  113. 113

    Meng, F. & Sachs, F. Visualizing dynamic cytoplasmic forces with a compliance-matched FRET sensor. J. Cell Sci. 124, 261–269 (2011).

    CAS  Article  Google Scholar 

  114. 114

    Wang, N., Tytell, J. D. & Ingber, D. E. Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus. Nature Rev. Mol. Cell Biol. 10, 75–82 (2009).

    CAS  Article  Google Scholar 

  115. 115

    Bustamante, C., Bryant, Z. & Smith, S. B. Ten years of tension: single-molecule DNA mechanics. Nature 421, 423–427 (2003).

    Article  CAS  Google Scholar 

  116. 116

    Greenleaf, W. J., Woodside, M. T. & Block, S. M. High-resolution, single-molecule measurements of biomolecular motion. Annu. Rev. Biophys. Biomol. Struct. 36, 171–190 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  117. 117

    Neuman, K. C. & Nagy, A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nature Methods 5, 491–505 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  118. 118

    Lipfert, J., Hao, X. & Dekker, N. H. Quantitative modeling and optimization of magnetic tweezers. Biophys. J. 96, 5040–5049 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  119. 119

    Strick, T. R., Allemand, J. F., Bensimon, D., Bensimon, A. & Croquette, V. The elasticity of a single supercoiled DNA molecule. Science 271, 1835–1837 (1996).

    CAS  Article  Google Scholar 

  120. 120

    van Oijen, A. M. & Loparo, J. J. Single-molecule studies of the replisome. Annu. Rev. Biophys. 39, 429–448 (2010).

    CAS  Article  Google Scholar 

  121. 121

    Butt, H. J., Cappella, B. & Kappl, M. Force measurements with the atomic force microscope: technique, interpretation and applications. Surf. Sci. Rep. 59, 1–152 (2005).

    CAS  Article  Google Scholar 

  122. 122

    Svoboda, K. & Block, S. M. Biological applications of optical forces. Annu. Rev. Biophys. Biomol. Struct. 23, 247–285 (1994).

    CAS  Article  Google Scholar 

  123. 123

    Moffitt, J. R., Chemla, Y. R., Smith, S. B. & Bustamante, C. Recent advances in optical tweezers. Annu. Rev. Biochem. 77, 205–228 (2008).

    CAS  Article  PubMed  Google Scholar 

  124. 124

    Greenleaf, W. J., Woodside, M. T., Abbondanzieri, E. A. & Block, S. M. Passive all-optical force clamp for high-resolution laser trapping. Phys. Rev. Lett. 95, 208102 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. 125

    van Aelst, K. et al. Type III restriction enzymes cleave DNA by long-range interaction between sites in both head-to-head and tail-to-tail inverted repeat. Proc. Natl Acad. Sci. USA 107, 9123–9128 (2010).

    CAS  Article  Google Scholar 

  126. 126

    Snapp, E. L. Fluorescent proteins: a cell biologist's user guide. Trends Cell Biol. 19, 649–655 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  127. 127

    Ramanathan, S. P. et al. Type III restriction enzymes communicate in 1D without looping between their target sites. Proc. Natl Acad. Sci. USA 106, 1748–1753 (2009).

    CAS  Article  Google Scholar 

  128. 128

    Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Traffic 2, 764–774 (2001).

    CAS  Article  Google Scholar 

  129. 129

    Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  130. 130

    Ram, S., Ward, E. S. & Ober, R. J. Beyond Rayleigh's criterion: a resolution measure with application to single-molecule microscopy. Proc. Natl Acad. Sci. USA 103, 4457–4462 (2006).

    CAS  Article  Google Scholar 

  131. 131

    Smith, C. S., Joseph, N., Rieger, B. & Lidke, K. A. Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nature Methods 7, 373–375 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  132. 132

    Mortensen, K. I., Churchman, L. S., Spudich, J. A. & Flyvbjerg, H. Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nature Methods 7, 377–381 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  133. 133

    Hell, S. W. Microscopy and its focal switch. Nature Methods 6, 24–32 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  134. 134

    Patterson, G., Davidson, M., Manley, S. & Lippincott-Schwartz, J. Superresolution imaging using single-molecule localization. Annu. Rev. Phys. Chem. 61, 345–367 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  135. 135

    Tu, L. C. & Musser, S. M. Single molecule studies of nucleocytoplasmic transport. Biochim. Biophys. Acta 1813, 1607–1618 (2011).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the European Science Foundation through a European Young Investigators (EURYI) grant to N.H.D. and by the Netherlands Organisation for Scientific Research through grants to N.H.D. and J.L. We thank anonymous referees for useful feedback and D. Grünwald for a critical reading of the manuscript. J. Kerssemakers is thanked for the visual layout of Figure 5. We acknowledge the many research efforts by groups in the field of genome processing and regret that owing to space limitations it was not possible to cite a larger number of high-quality works.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Nynke H. Dekker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

Related links

FURTHER INFORMATION

Nynke H. Dekker's homepage

Glossary

Replisome

A multi-protein complex that carries out DNA replication.

Tethered particle motion

(TPM). A single-molecule technique that uses tethered beads to study biological molecules in the absence of any externally applied force. Changes in the average position of the bead can report on changes in tether length and hence on enzyme activity.

Evanescent waves

Parallel optical waves with an exponentially decaying intensity that occur near a surface when incident light impinges at an angle greater than the critical angle for refraction.

Rayleigh criterion

Quantifies the minimum resolvable distance between two objects that fluoresce at the same wavelength. This distance equals roughly half the wavelength of light.

RNA polymerase holoenzyme

The initiation complex composed of the RNA polymerase core enzyme and the σ-initiation factor.

σ54

One of the initiation factors that can bind to Escherichia coli RNA polymerase during initiation to allow it to recognize a specific promoter sequence.

σ70

The most common and most widely studied initiation factor that can bind to Escherichia coli RNA polymerase during initiation to allow it to recognize a specific promoter sequence.

Elongation factor G

(EF-G). A factor that provides the bacterial ribosome with the necessary energy (derived from GTP hydrolysis) required to translocate along the mRNA.

Ribosomal A, P and E sites

The aminoacyl, peptidyl and exit sites of the ribosome, respectively, which are the three different binding sites of tRNAs.

Nucleoporins

Proteins that comprise the nuclear pore complex.

Super-registration microscopy

The use of fluorescence microscopy to localize fluorescent probes as described in Box 2 in a way that accurately registers the relative positions of labels that fluoresce in different colours.

Mechanochemical

A description of how the chemical reactions driving biological processes, such as ATP hydrolysis in a molecular motor, are coupled to mechanical motion: for example, translocation along a nucleic acid template.

Primosome

A protein complex consisting of a helicase and primase that is responsible for the synthesis of RNA primers during DNA replication.

RNA aptamers

RNA molecules that specifically bind to a target molecule.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Dulin, D., Lipfert, J., Moolman, M. et al. Studying genomic processes at the single-molecule level: introducing the tools and applications. Nat Rev Genet 14, 9–22 (2013). https://doi.org/10.1038/nrg3316

Download citation

Further reading