Biological mechanisms, one molecule at a time.

The last 15 years have witnessed the development of tools that allow the observation and manipulation of single molecules. The rapidly expanding application of these technologies for investigating biological systems of ever-increasing complexity is revolutionizing our ability to probe the mechanisms of biological reactions. Here, we compare the mechanistic information available from single-molecule experiments with the information typically obtained from ensemble studies and show how these two experimental approaches interface with each other. We next present a basic overview of the toolkit for observing and manipulating biology one molecule at a time. We close by presenting a case study demonstrating the impact that single-molecule approaches have had on our understanding of one of life's most fundamental biochemical reactions: the translation of a messenger RNA into its encoded protein by the ribosome.

[1]  Th. Förster Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[2]  R. Benesch,et al.  Enzymatic removal of oxygen for polarography and related methods. , 1953, Science.

[3]  B. Rotman,et al.  Measurement of activity of single molecules of beta-D-galactosidase. , 1961, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Pestka Studies on the formation of transfer ribonucleic acid-ribosome complexes. VI. Oligopeptide synthesis and translocation on ribosomes in the presence and absence of soluble transfer factors. , 1969, The Journal of biological chemistry.

[5]  S. Pestka Studies on the formation of transfer ribonucleic acid-ribosome complexes. IX. Effect of antibiotics on translocation and peptide bond formation. , 1970, Archives of biochemistry and biophysics.

[6]  A. Ashkin Acceleration and trapping of particles by radiation pressure , 1970 .

[7]  S. Pestka Studies on the formation of transfer ribonucleic acid-ribosome complexes. 8. Survey of the effect of antibiotics of N-acetyl-phenylalanyl-puromycin formation: possible mechanism of chloramphenicol action. , 1970, Archives of biochemistry and biophysics.

[8]  A. Spirin,et al.  Factor-free ("non-enzymic") and factor-dependent systems of translation of polyuridylic acid by Escherichia coli ribosomes. , 1976, Journal of molecular biology.

[9]  T. Hirschfeld Optical microscopic observation of single small molecules. , 1976, Applied optics.

[10]  D. Axelrod Cell-substrate contacts illuminated by total internal reflection fluorescence , 1981, The Journal of cell biology.

[11]  K. Nierhaus,et al.  Spontaneous, elongation factor G independent translocation of Escherichia coli ribosomes. , 1983, The Journal of biological chemistry.

[12]  N. Thompson,et al.  Total internal reflection fluorescence. , 1984, Annual review of biophysics and bioengineering.

[13]  Gerber,et al.  Atomic Force Microscope , 2020, Definitions.

[14]  R. Keller,et al.  Ultrasensitive laser-induced fluorescence detection in hydrodynamically focused flows , 1987 .

[15]  R A Mathies,et al.  Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[16]  W. Moerner,et al.  Optical detection and spectroscopy of single molecules in a solid. , 1989, Physical review letters.

[17]  Harry F. Noller,et al.  Intermediate states in the movement of transfer RNA in the ribosome , 1989, Nature.

[18]  R. Weiss,et al.  Ribosome gymnastics—Degree of difficulty 9.5, style 10.0 , 1990, Cell.

[19]  M. Orrit,et al.  Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal. , 1990, Physical review letters.

[20]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.

[21]  Rudolf Rigler,et al.  Diffusion of single molecules through a Gaussian laser beam , 1993, Other Conferences.

[22]  Robert J. Chichester,et al.  Single Molecules Observed by Near-Field Scanning Optical Microscopy , 1993, Science.

[23]  D E Smith,et al.  Direct observation of tube-like motion of a single polymer chain. , 1994, Science.

[24]  M. Rodnina,et al.  Transient conformational states of aminoacyl-tRNA during ribosome binding catalyzed by elongation factor Tu. , 1994, Biochemistry.

[25]  Xie,et al.  Single molecule emission characteristics in near-field microscopy. , 1995, Physical review letters.

[26]  Kiwamu Saito,et al.  Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution , 1995, Nature.

[27]  T M Jovin,et al.  Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Louis E. Brus,et al.  Imaging and Time-Resolved Spectroscopy of Single Molecules at an Interface , 1996, Science.

[29]  D. F. Ogletree,et al.  Probing the interaction between single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor , 1996, Summaries of Papers Presented at the Quantum Electronics and Laser Science Conference.

[30]  R. Brimacombe,et al.  Visualization of elongation factor Tu on the Escherichia coli ribosome , 1997, Nature.

[31]  T. Pape,et al.  Complete kinetic mechanism of elongation factor Tu‐dependent binding of aminoacyl‐tRNA to the A site of the E.coli ribosome , 1998, The EMBO journal.

[32]  H. Noller,et al.  EF‐G‐catalyzed translocation of anticodon stem–loop analogs of transfer RNA in the ribosome , 1998, The EMBO journal.

[33]  X. Zhuang,et al.  Ligand-induced conformational changes observed in single RNA molecules. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[34]  S. Weiss Fluorescence spectroscopy of single biomolecules. , 1999, Science.

[35]  Arthur Ashkin,et al.  Optical Trapping and Manipulation of Neutral Particles Using Lasers , 1999 .

[36]  Magnetic resonance of a single molecular spin , 1999 .

[37]  Joachim Frank,et al.  A ratchet-like inter-subunit reorganization of the ribosome during translocation , 2000, Nature.

[38]  H Li,et al.  Atomic force microscopy reveals the mechanical design of a modular protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[39]  D. Ballou,et al.  The use of protocatechuate dioxygenase for maintaining anaerobic conditions in biochemical experiments. , 2000, Analytical biochemistry.

[40]  Alois Renn,et al.  Direct observation of the triplet lifetime quenching of single dye molecules by molecular oxygen , 2001 .

[41]  G. Haran,et al.  Immobilization in Surface-Tethered Lipid Vesicles as a New Tool for Single Biomolecule Spectroscopy , 2001 .

[42]  T. Ha,et al.  Single-molecule fluorescence resonance energy transfer. , 2001, Methods.

[43]  J. Liphardt,et al.  Reversible Unfolding of Single RNA Molecules by Mechanical Force , 2001, Science.

[44]  D. Axelrod Total internal reflection fluorescence microscopy in cell biology. , 2003, Methods in enzymology.

[45]  Joachim Frank,et al.  Cryo‐EM reveals an active role for aminoacyl‐tRNA in the accommodation process , 2002, The EMBO journal.

[46]  M. Heel,et al.  Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex , 2002, Nature Structural Biology.

[47]  I. Tinoco,et al.  Equilibrium Information from Nonequilibrium Measurements in an Experimental Test of Jarzynski's Equality , 2002, Science.

[48]  Nancy R. Forde,et al.  Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Mark E. Welland,et al.  Electronic spin detection in molecules using scanning-tunneling- microscopy-assisted electron-spin resonance , 2002 .

[50]  F. Ritort,et al.  A two-state kinetic model for the unfolding of single molecules by mechanical force , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[51]  Taekjip Ha,et al.  Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase , 2002, Nature.

[52]  Ignacio Tinoco,et al.  The effect of force on thermodynamics and kinetics of single molecule reactions. , 2002, Biophysical chemistry.

[53]  A. Knight,et al.  Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy. , 2003, Methods.

[54]  Carlos Bustamante,et al.  Optical-trap force transducer that operates by direct measurement of light momentum. , 2003, Methods in enzymology.

[55]  Scott M Stagg,et al.  Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy , 2003, Nature Structural Biology.

[56]  Michael D. Stone,et al.  Chirality sensing by Escherichia coli topoisomerase IV and the mechanism of type II topoisomerases , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[57]  S. Turner,et al.  Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations , 2003, Science.

[58]  Michael D. Stone,et al.  Structural transitions and elasticity from torque measurements on DNA , 2003, Nature.

[59]  Polly M Fordyce,et al.  Simultaneous, coincident optical trapping and single-molecule fluorescence , 2004, Nature Methods.

[60]  J. Puglisi,et al.  tRNA selection and kinetic proofreading in translation , 2004, Nature Structural &Molecular Biology.

[61]  Nancy R Forde,et al.  Mechanical processes in biochemistry. , 2004, Annual review of biochemistry.

[62]  Taekjip Ha,et al.  Single-molecule three-color FRET. , 2004, Biophysical journal.

[63]  Ignacio Tinoco,et al.  Force as a useful variable in reactions: unfolding RNA. , 2004, Annual review of biophysics and biomolecular structure.

[64]  Steven Chu,et al.  tRNA dynamics on the ribosome during translation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[65]  J. Holton,et al.  Structures of the Bacterial Ribosome at 3.5 Å Resolution , 2005, Science.

[66]  C. Jarzynski,et al.  Verification of the Crooks fluctuation theorem and recovery of RNA folding free energies , 2005, Nature.

[67]  R. Kołos,et al.  Optimal oxygen concentration for the detection of single indocarbocyanine molecules in a polymeric matrix , 2005 .

[68]  Taekjip Ha,et al.  Surfaces and orientations: much to FRET about? , 2004, Accounts of chemical research.

[69]  Nam Ki Lee,et al.  Alternating‐Laser Excitation of Single Molecules , 2005 .

[70]  Helmut Grubmüller,et al.  Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter FRET measurements. , 2005, RNA.

[71]  Hector H. Huang,et al.  Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[72]  X. Xie,et al.  Probing Gene Expression in Live Cells, One Protein Molecule at a Time , 2006, Science.

[73]  M. Rodnina,et al.  The ribosome's response to codon-anticodon mismatches. , 2006, Biochimie.

[74]  Ignacio Tinoco,et al.  Probing the mechanical folding kinetics of TAR RNA by hopping, force-jump, and force-ramp methods. , 2006, Biophysical journal.

[75]  Divya Sharma,et al.  The hybrid state of tRNA binding is an authentic translation elongation intermediate , 2006, Nature Structural &Molecular Biology.

[76]  M. Rodnina,et al.  Mechanism of peptide bond formation on the ribosome , 2006, Quarterly Reviews of Biophysics.

[77]  V. Sandoghdar,et al.  Oxygen-dependent photochemistry of fluorescent dyes studied at the single molecule level , 2006 .

[78]  Ignacio Tinoco,et al.  Determination of thermodynamics and kinetics of RNA reactions by force , 2006, Quarterly Reviews of Biophysics.

[79]  N. Friedman,et al.  Stochastic protein expression in individual cells at the single molecule level , 2006, Nature.

[80]  J. Puglisi,et al.  Thiostrepton inhibition of tRNA delivery to the ribosome. , 2007, RNA.

[81]  Harry F Noller,et al.  Intersubunit movement is required for ribosomal translocation , 2007, Proceedings of the National Academy of Sciences.

[82]  Julio M Fernandez,et al.  Dwell-time distribution analysis of polyprotein unfolding using force-clamp spectroscopy. , 2007, Biophysical journal.

[83]  F. Ritort,et al.  Force unfolding kinetics of RNA using optical tweezers. II. Modeling experiments. , 2007, Biophysical journal.

[84]  Wolfgang Wintermeyer,et al.  How ribosomes make peptide bonds. , 2007, Trends in biochemical sciences.

[85]  J. Puglisi,et al.  The role of fluctuations in tRNA selection by the ribosome , 2007, Proceedings of the National Academy of Sciences.

[86]  Ignacio Tinoco,et al.  Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results. , 2007, Biophysical journal.

[87]  J. Doudna,et al.  Quantitative studies of ribosome conformational dynamics , 2007, Quarterly Reviews of Biophysics.

[88]  Joachim Frank,et al.  The process of mRNA–tRNA translocation , 2007, Proceedings of the National Academy of Sciences.

[89]  Carlos Bustamante,et al.  Backtracking determines the force sensitivity of RNAP II in a factor-dependent manner , 2007, Nature.

[90]  Sotaro Uemura,et al.  [Imaging and nano-manipulation of single biomolecules]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[91]  Steven Chu,et al.  Fluctuations of transfer RNAs between classical and hybrid states. , 2007, Biophysical journal.

[92]  Takuya Terai,et al.  Fluorescent probes for bioimaging applications. , 2008, Current opinion in chemical biology.

[93]  Rahul Roy,et al.  A practical guide to single-molecule FRET , 2008, Nature Methods.

[94]  P. Schultz,et al.  A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer. , 2008, Journal of the American Chemical Society.

[95]  Carlos Bustamante,et al.  In singulo biochemistry: when less is more. , 2008, Annual review of biochemistry.

[96]  Peiqian Zhao,et al.  Parallel confocal detection of single molecules in real time. , 2008, Optics letters.

[97]  Colin Echeverría Aitken,et al.  An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. , 2008, Biophysical journal.

[98]  Ignacio Tinoco,et al.  Following translation by single ribosomes one codon at a time , 2008, Nature.

[99]  Paul R. Selvin,et al.  Single-molecule techniques : a laboratory manual , 2008 .

[100]  Anthony J. Manzo,et al.  Do-it-yourself guide: how to use the modern single-molecule toolkit , 2008, Nature Methods.

[101]  H. Craighead,et al.  Zero-mode waveguides: sub-wavelength nanostructures for single molecule studies at high concentrations. , 2008, Methods.

[102]  Ignacio Tinoco,et al.  How RNA unfolds and refolds. , 2008, Annual review of biochemistry.

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

[104]  Joseph D. Puglisi,et al.  Irreversible chemical steps control intersubunit dynamics during translation , 2008, Proceedings of the National Academy of Sciences.

[105]  Taekjip Ha,et al.  Spontaneous intersubunit rotation in single ribosomes. , 2008, Molecular cell.

[106]  M. Moore,et al.  Visualizing the splicing of single pre-mRNA molecules in whole cell extract. , 2007, RNA.

[107]  Carlos Bustamante,et al.  Recent advances in optical tweezers. , 2008, Annual review of biochemistry.

[108]  I. Tinoco,et al.  Simulation and analysis of single-ribosome translation , 2009, Physical biology.

[109]  T. Leyh,et al.  Natural amino acids do not require their native tRNAs for efficient selection by the ribosome , 2009, Nature chemical biology.

[110]  Colin Echeverría Aitken,et al.  GTP hydrolysis by IF2 guides progression of the ribosome into elongation. , 2009, Molecular cell.

[111]  James B. Munro,et al.  Mitigating unwanted photophysical processes for improved single-molecule fluorescence imaging. , 2009, Biophysical journal.

[112]  Taekjip Ha,et al.  Following movement of the L1 stalk between three functional states in single ribosomes , 2009, Proceedings of the National Academy of Sciences.

[113]  David Rueda,et al.  Single Molecule Analysis of Protein Free U2/U6 snRNAs , 2009, Nature Structural &Molecular Biology.

[114]  Ruben L. Gonzalez,et al.  Coupling of Ribosomal L1 Stalk and tRNA Dynamics during Translation Elongation , 2009 .

[115]  Wei Zhang,et al.  GTPase activation of elongation factor EF‐Tu by the ribosome during decoding , 2009, The EMBO journal.

[116]  Sarah E. Walker,et al.  Ribosomal translocation: one step closer to the molecular mechanism. , 2009, ACS chemical biology.

[117]  Peter Hinterdorfer,et al.  Handbook of single-molecule biophysics , 2009 .

[118]  James B. Munro,et al.  Spontaneous formation of the unlocked state of the ribosome is a multistep process , 2009, Proceedings of the National Academy of Sciences.

[119]  S. Turner,et al.  Real-Time DNA Sequencing from Single Polymerase Molecules , 2009, Science.

[120]  J. Cate,et al.  Structures of the Ribosome in Intermediate States of Ratcheting , 2009, Science.

[121]  Jake M. Hofman,et al.  Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation , 2009, Proceedings of the National Academy of Sciences.

[122]  Wolfgang Wintermeyer,et al.  Recent mechanistic insights into eukaryotic ribosomes. , 2009, Current opinion in cell biology.

[123]  Klaus Schulten,et al.  Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis , 2009, Proceedings of the National Academy of Sciences.

[124]  H. Chapman,et al.  X-ray imaging beyond the limits. , 2009, Nature materials.

[125]  S. Blanchard Single-molecule observations of ribosome function. , 2009, Current opinion in structural biology.

[126]  Yong-Gui Gao,et al.  The Crystal Structure of the Ribosome Bound to EF-Tu and Aminoacyl-tRNA , 2009, Science.

[127]  Paul V. Ruijgrok,et al.  Room-Temperature Detection of a Single Molecule’s Absorption by Photothermal Contrast , 2010, Science.

[128]  Taekjip Ha,et al.  Single-molecule four-color FRET. , 2010, Angewandte Chemie.

[129]  Joseph D. Puglisi,et al.  Following the intersubunit conformation of the ribosome during translation in real time , 2010, Nature Structural &Molecular Biology.

[130]  Alois Renn,et al.  Single-Molecule Sensitivity in Optical Absorption at Room Temperature , 2010 .

[131]  Joachim Frank,et al.  Structure and dynamics of a processive Brownian motor: the translating ribosome. , 2010, Annual review of biochemistry.

[132]  James B. Munro,et al.  Correlated conformational events in EF-G and the ribosome regulate translocation , 2010, Nature Structural &Molecular Biology.

[133]  Soojin Lim,et al.  NIR dyes for bioimaging applications. , 2010, Current opinion in chemical biology.

[134]  Michael B. Feldman,et al.  Conformational sampling of aminoacyl-tRNA during selection on the bacterial ribosome. , 2010, Journal of molecular biology.

[135]  X. Zhuang,et al.  Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells , 2010, Cell.

[136]  M. Yusupov,et al.  Crystal Structure of the Eukaryotic Ribosome , 2010, Science.

[137]  Tae-Hee Lee,et al.  Codon-dependent tRNA fluctuations monitored with fluorescence polarization. , 2010, Biophysical journal.

[138]  Wei Min,et al.  Ground-State Depletion Microscopy: Detection Sensitivity of Single-Molecule Optical Absorption at Room Temperature , 2010 .

[139]  Paul J. Choi,et al.  Quantifying E. coli Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells , 2010, Science.

[140]  A. Kelley,et al.  The Mechanism for Activation of GTP Hydrolysis on the Ribosome , 2010, Science.

[141]  C. Guthrie,et al.  Conformational dynamics of single pre-mRNA molecules during in vitro splicing , 2010, Nature Structural &Molecular Biology.

[142]  Yuhong Wang,et al.  Single-molecule study of viomycin's inhibition mechanism on ribosome translocation. , 2010, Biochemistry.

[143]  Elizabeth A. Shank,et al.  The folding cooperativity of a protein is controlled by its chain topology , 2010, Nature.

[144]  James B. Munro,et al.  A fast dynamic mode of the EF‐G‐bound ribosome , 2010, The EMBO journal.

[145]  Colin Echeverría Aitken,et al.  Real-time tRNA transit on single translating ribosomes at codon resolution , 2010, Nature.

[146]  N. Tanner,et al.  Visualizing DNA replication at the single-molecule level. , 2010, Methods in enzymology.

[147]  Michael B. Feldman,et al.  Aminoglycoside activity observed on single pre-translocation ribosome complexes. , 2010, Nature chemical biology.

[148]  Joseph D Puglisi,et al.  Single ribosome dynamics and the mechanism of translation. , 2010, Annual review of biophysics.

[149]  S. Hell Far-field optical nanoscopy , 2010 .

[150]  T. Ha,et al.  Force-fluorescence spectroscopy at the single-molecule level. , 2010, Methods in enzymology.

[151]  Zygmunt Gryczynski,et al.  Enhancement of single-molecule fluorescence signals by colloidal silver nanoparticles in studies of protein translation. , 2011, ACS nano.

[152]  T. Ha,et al.  Forcing a connection: Impacts of single‐molecule force spectroscopy on in vivo tension sensing , 2011, Biopolymers.

[153]  Jonathan E. Bronson,et al.  Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis , 2011, Nature Structural &Molecular Biology.

[154]  Ignacio Tinoco,et al.  The Ribosome Uses Two Active Mechanisms to Unwind mRNA During Translation , 2011, Nature.

[155]  Bin Wang,et al.  A microfluidic approach for investigating the temperature dependence of biomolecular activity with single-molecule resolution. , 2011, Lab on a chip.

[156]  A. Revyakin,et al.  Single-molecule studies using magnetic traps. , 2012, Cold Spring Harbor protocols.

[157]  C. Joo,et al.  Single-molecule FRET with total internal reflection microscopy. , 2012, Cold Spring Harbor protocols.