Extreme bendability of DNA less than 100 base pairs long revealed by single-molecule cyclization

Abstract

The classical view of DNA posits that DNA must be stiff below the persistence length [<150 base pairs (bp)], but recent studies addressing this have yielded contradictory results. We developed a fluorescence-based, protein-free assay for studying the cyclization of single DNA molecules in real time. The assay samples the equilibrium population of a sharply bent, transient species that is entirely suppressed in single-molecule mechanical measurements and is biologically more relevant than the annealed species sampled in the traditional ligase-based assay. The looping rate has a weak length dependence between 67 and 106 bp that cannot be described by the worm-like chain model. Many biologically important protein-DNA interactions that involve looping and bending of DNA below 100 bp likely use this intrinsic bendability of DNA.

Figure 1

(A) Donor (Cy3) and acceptor (Cy5) labeled DNA molecules were immobilized on the surface via biotin-neutravidin interaction. (B) Fluorescence images of single 91 bp DNA molecules in corresponding donor and acceptor channels are shown before (left panels) and 20 minutes after adding high salt (1 M NaCl) buffer (right panels). Scale bar is 5 μm. (C) Histograms of FRET efficiency as a function of time (t=0 is when high salt was introduced) show the evolution of looped (high FRET) and unlooped (low FRET) populations. (D) Fraction of looped DNA (high FRET population) as a function of time, measured from the histograms in C. An exponential fit to this curve gives the looping rate R. Data are means ± SEM (N = 5).

Figure 2

(A) Looping time as a function of DNA circular length for surface tethered DNA (black squares) and vesicle encapsulated DNA molecules (red squares). (B) Looping time for 7 DNA sequences with 63 basepair duplex length and 10 nt overhang. R73 is the standard sequence used in A. Poly-A constructs were constructed by inserting n=10, 17, 26 and 38 consecutive A bases in the middle of a random sequence (E8). Data are means ± SEM (N≥3).

Figure 3

(A) Representative fluorescence intensities (top, green for donor and red for acceptor) and corresponding FRET efficiency (bottom, blue) time traces measured from a single DNA molecule in 750 mM NaCl. The DNA has 91 bp initial dsDNA with 8 nt single stranded overhangs. The arrow indicates a direct acceptor excitation to verify the acceptor has not photobleached. (B) Looping and unlooping rates as a function of [NaCl]. The DNA has 91 bp initial dsDNA with 8 nt ss-overhangs. (C) Bimolecular association rate (k_on) measured as shown in Fig. s2 shows the same 3 fold increase as the looping rate with increasing [NaCl]. Data are means ± SEM (N≥300 molecules). (D) The model to relate looping rate (R), bimolecular association rate (k_on) and apparent j-factor. (E) j-factor for surfaced tethered DNA (black squares) and vesicle encapsulated DNA (red circles). (F) Measured j-factor for surface tethered DNA (black squares) and vesicle encapsulated DNA (red squares). Solid black curve is the Shimada-Yamakawa prediction for DNA cyclization. Dashed line and dotted line are the WLC predictions for the j-factor of DNA circles with free boundary condition and for DNA molecules with 5 nm capture radius, respectively. Data are means ± SEM (N≥3).

Figure 4

(A) Our assay reports on the equilibrium population of the intermediate state (dashed box) with the two DNA ends in close proximity. (B) Schematic representation of DNA cyclization reaction steps in the ligase assay. The intermediate state with the two DNA ends in close proximity (solid box) does not get sampled in this assay. Instead, the ligase samples the equilibrium population of the annealed state (dashed box). Ligase protein is labeled L.