1. Single-Molecule Biology: What Is It and How Does It Work?
Jordanka Zlatanova, Kensal van Holde 
Molecular Cell 
Volume 24, Issue 3, Pages (November 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Detecting Fluorescence from Individual Fluorophores by Total Internal Reflection Fluorescence Microscopy and Principle of Single-Pair FRET for Studying Biological Macromolecules
(A) In total internal reflection fluorescence (TIRF) microscopes, the sample is excited by the intense electromagnetic field that is created by the totally reflected laser beam at the interface of two media of different refractive indexes (usually quartz and water). This field, also known as an evanescent wave, decays exponentially with the distance from the interface and thus excites only the fluorophores present in a small volume in close proximity to the quartz. The figure illustrates the more commonly used prism-based setup; the advantages of the alternative, objective-type TIRFM are discussed in Knight et al. (2005).
(B) Principle of spFRET (see text).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Use of the Atomic Force Microscope for Imaging and Force Spectroscopy
(A) Schematic of the principle of action of the AFM (see text).
(B) A schematic representation showing the structural transitions in a multidomain protein upon mechanical stretching using the AFM. The sawtooth pattern of the force-extension curves results from strings of successive enthalpic and entropic portions, reflecting the unfolding of individual domains, followed by entropic stretching of the unfolded domain. The unfolding of each domain adds significant length to the chain and relaxes the stress on the cantilever, which returns to its nondeflected state. The unfolded portion of the polypeptide chain can now undergo entropic stretching, which is accompanied by gradual deflection of the cantilever (adapted from Zlatanova et al. [2000]).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Optical Trapping and Its Application for Stretching DNA and Chromatin Fibers
(A) The behavior of a dielectric bead in a focused laser beam (see text).
(B) OT can be used to study macromolecules by attaching them to the optically trapped bead at one end and to a movable platform (in the illustration, another bead held in a micropipette) at the other end. Moving the platform away from the trapped bead will apply stretching forces to the macromolecule, causing displacement of the trapped bead from its equilibrium position. This displacement can be measured and used to estimate the forces applied to the molecule.
(C) Stretching of λ-DNA. A typical DNA force-extension curve exhibits three portions: entropic stretching of the worm-like chain at the beginning, enthalpic (elastic) stretching of the structure at lengths of the molecule approaching or slightly exceeding the contour length (in this case ∼16 μm), and finally, at ∼65 pN force, a transition from the B to the so-called S (stretched) form. The physical nature of the S form has been controversial (for detailed discussion, see Bustamante et al. [2000b, 2003]; Zlatanova and Leuba [2002, 2003a, 2004]). The DNA was stretched to ∼20 μm (red curve) and then relaxed (blue curve).
(D) Stretching of a chromatin fiber assembled on naked λ-DNA molecule by the addition of X. laevis egg extract directly into the flow cell of the instrument. The extract contains core histones and protein factors needed for assembly (assembly is manifested by shortening of the distance between the two beads with time). Note the sharp discontinuities in the force-extension curve reflecting the unraveling of the DNA from around the histone octamer that forms the core of the nucleosomal particles. Nucleosomes can unwrap either individually or in groups of two, three, or four. At high extension, when all histones have been forced off, the curve approaches that of naked DNA. Note that the two force-extension curves for DNA (C) and chromatin (D) are aligned with respect to the length of the structure during stretching, so that a direct comparison of the behavior of DNA and of chromatin is possible.
(C) and (D) are modified with author permission (Bennink, 2001).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Magnetic Tweezers and Their Application for Studying the Response of DNA to Torsion
(A) Schematic of the principle of action of MT (see text).
(B) Relative extension of a DNA molecule as a function of the degree of imposed supercoiling (reproduced from Strick et al. [1998], with permission from the Biophysical Society; the figure has been slightly modified in terms of the labeling of the lines). MT were used to introduce a controlled level of supercoiling density in a single molecule of DNA tethered between a surface and a superparamagnetic bead in a topologically constrained way. The changes in tether length (extension) were followed as the external magnets were rotated either clockwise or counterclockwise, to create negative or positive supercoiling tension, respectively. Force-extension curves taken at different levels of stretching forces reveal intriguing differences in the behavior of positively versus negatively supercoiled DNA. The linear shortening of the molecules results from plectoneme formation, whereas the portions of the curves that remain parallel to the x axis despite the continuous pumping in of superhelical tension reflect structural transitions in the molecules that are not accompanied by shortening. In the case of negative superhelical tension, the helix undergoes local denaturation (Strick et al., 1998). In the case of positive superhelical tension, the B-DNA undergoes a structural transition to a new phase called P-DNA (Pauling-DNA, Allemand et al. [1998]); in this conformation, the phosphate-sugar backbones of the two strands are wound inside the structure, with the bases exposed to the solution.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Studying Transcription Elongation
(A) Schematic of the tethered particle motion (TPM) method, based on the dependence of the extent of Brownian motion of a bead (readily measured by light microscopy) on the length of the DNA template tethering it to the surface. In transcription experiments, the polymerase is immobilized on a glass surface and a bead is attached to either the upstream or the downstream end of the DNA template; elongation will result in lengthening or shortening of the tether (and a change in the Brownian motion of the bead), depending on the particular geometry used.
(B) Transcription elongation as measured by the TPM method. Example tether-length/time courses with the bead at the upstream (left traces) or downstream (right traces) end of the template (reproduced from Yin et al. [1994] with permission from the Biophysical Society).
(C) Concept of recent experiments from S. Block's laboratory using the novel passive, all-optical force clamp that takes advantage of the existence of a region in the trap where the force is approximately constant for small bead displacements (Greenleaf et al., 2005; Herbert et al., 2006). Two beads are held in separate optical traps: the transcribing polymerase is attached to one of the beads and the upstream end of DNA to the other bead, creating a bead-DNA-RNAP-bead dumbbell geometry. In the configuration used, the trap applies an assisting load. This instrument achieves angstrom-level resolution.
(D) Representative records of transcription along a repetitive DNA template containing eight repeats of an ∼230 bp sequence that possesses previously identified pause sites. Most records display distinct pauses at the expected pause sites; in addition, other pause sites are identified that display sequence similarities (reprinted from Herbert et al. [2006] with permission from Cell Press).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Representative SM Data on Kinesin Moving Along Microtubules
(A) Experimental geometry for observing stalk rotation: a microtubule is bound to a single-surface-attached kinesin molecule.
(B) In the presence of ATP, there is no rotation of the stalk, as seen by the overlaid traces (arrows) of a microtubule imaged at 1 s intervals. The right- and the left-hand traces show the same microtubule in two different time periods separated by 104 s (adapted from Hua et al. [2002] and reproduced with permission from AAAS).
(C) Time course of the displacement of a single heterodimeric kinesin molecule, as visualized by OT nanometry (three left traces). This molecule contains two heads with very different mechanochemical cycle rates (wild-type and a single point mutant in the nucleotide-binding motif, with much-reduced microtubule-gliding speed). The traces indicate that in most cases the steps are 16 nm, but sometimes a shoulder at 8 nm is distinguishable. Thus, the observed 16 nm step is actually two successive steps of very different dwell times. The wild-type homodimer (two right traces) shows clear 8 nm steps of almost equal duration (reproduced from Kaseda et al. [2003] with permission from Nature Publishing Group, McMillan Publishers Ltd.).
(D) Stepwise motion of recombinant kinesin homodimers as measured by OT: intrinsic stepping rate can alternate between two different values at each sequential step, causing the molecule to limp. The construct tested was DmK401, a truncated derivative of D. melanogaster kinesin containing two identical heads and a sufficient length of the neck coiled coil for dimerization. The dwell times between successive 8 nm steps alternate between slow and fast phases, causing steps to appear in pairs (red and blue). The dwell times in the record are numbered sequentially to make the point that the longer-lived dwell times tend to cluster systematically, in this specific trace, in the odd-numbered subset. The vertical bars indicate the occurrence of the steps (traces reproduced from Asbury et al. [2003], with permission from AAAS).
(E) A typical image of surface-immobilized Cy3-DNA acquired within 0.5 s using objective-type TIRF. The image contains ∼14,000 photons and can be fitted to a two-dimensional Gaussian, which approximates the point spread function (PSF) of the Cy3; the center of the PSF yields ∼1 nm precision and accuracy in the center localization of the fluorescence spot (reproduced from Yildiz and Selvin [2005a] with permission from the American Chemical Society). This technique has been named FIONA (Yildiz et al., 2003; Yildiz and Selvin, 2005a).
(F) Position versus time for kinesin motility as determined by FIONA: example traces for an E215C homodimer kinesin. Dots, experimental points; red lines show when steps occur (vertical jumps) and the average positions between steps (plateaus). Both the step sizes of an individual head of a kinesin dimer and dwell-time analysis support a hand-over-hand mechanism (reproduced from Yildiz et al. [2004] with permission from AAAS).
Molecular Cell  , DOI: ( /j.molcel )
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