Improving signal/noise resolution in single-molecule experiments using molecular constructs with short handles - PubMed (original) (raw)

Improving signal/noise resolution in single-molecule experiments using molecular constructs with short handles

N Forns et al. Biophys J. 2011.

Abstract

We investigate unfolding/folding force kinetics in DNA hairpins exhibiting two and three states with newly designed short dsDNA handles (29 bp) using optical tweezers. We show how the higher stiffness of the molecular setup moderately enhances the signal/noise ratio (SNR) in hopping experiments as compared to conventional long-handled constructs (≅700 bp). The shorter construct results in a signal of higher SNR and slower folding/unfolding kinetics, thereby facilitating the detection of otherwise fast structural transitions. A novel analysis, as far as we are aware, of the elastic properties of the molecular setup, based on high-bandwidth measurements of force fluctuations along the folded branch, reveals that the highest SNR that can be achieved with short handles is potentially limited by the marked reduction of the effective persistence length and stretch modulus of the short linker complex.

PubMed Disclaimer

Figures

Figure 1

Experimental setup. (A) The molecular construct is attached between two beads, one held by the suction of a micropipette and the other captured in the optical trap. (B) Molecular construct with dsDNA short handles (29 bp/handle) made of three different oligonucleotides. (C and D) PM (C) and CFM (D) time-dependent trap-pipette relative distances (upper) and time-dependent force traces (lower) for the 2S hairpin with short handles. (E) PM time-dependent force trace (upper) and CFM time-dependent relative distances (lower) for the 3S hairpin with short handles.

Figure 2

2S hairpin. (A) Sequence of the hairpin. (B) Free-energy landscape plotted as a function of the molecular extension (nm) at force f = 14.6 pN (at 25°C and 1 M NaCl). This was computed as described in Section S1. We also indicate the different transition rates (arrows). (C) FDC of pulling experiments with long and short handles. (Insets) Unfolding and refolding of the hairpin. (D and E) Force traces and distributions in the PM experiments for short- (upper) and long-handled (lower) constructs. (F) Time-dependent relative distances in the CFM experiments for the short- (upper) and long-handled (lower) constructs.

Figure 3

3S hairpin. (A) Sequence of the hairpin. (B) Free-energy landscape plotted as a function of the molecular extension (nm) at force f = 14.1 pN (at 25°C and 1 M NaCl). This was computed as described in Section S1. We also indicate the different transition rates (arrows). (C) FDC of pulling experiments with long and short handles. (Insets) Unfolding and refolding of the hairpin. (D and E) Force traces and their distribution in the PM experiments for the short- (upper) and long-handled constructs (lower). (F) Time-dependent relative distances in the CFM experiments for the short- (upper) and long-handled (lower) constructs.

Figure 4

Results for the 2S hairpin with short handles (red or dark gray) and long handles (blue or light gray) (color online). (A and B) Plots of k as a function of force for CFM experiments (A) and PM experiments (B) and the linear fit (lines) for the log of the rates, where solid symbols and lines represent the unfolding rate (kFU) and dotted symbols and lines the folding rate (kUF). (C and D) Plots of the ΔGFU versus force for CFM (C) and PM (D) experiments and their linear fit (lines). We show the mean values for each point and the standard error. Molecule statistics are presented in the note to Table 1.

Figure 5

Results for the 3S hairpins with short handles (A) and long handles (B). Plots of k as a function of force for the CFM (upper) and PM (middle) experiments. Linear fits to the log of the rates are shown, with unfolding rates (kFI and kIU) indicated by solid lines and symbols and folding rates (kIF and kUI) by dotted lines and symbols. (Lower) Δ_GFI_ (blue or light gray) and Δ_GUI_ (red or dark gray) (color online) versus force for PM (solid circles) and CFM (open circles). Linear fits are shown as solid lines for PM and dotted lines for CFM. We show the mean values for each point and the to error. Molecule statistics are presented in the note of Table 1.

Figure 6

Analysis of force variance and stiffness (1–15 pN) for the 2S hairpin. (A) Typical force traces (at ≅13 pN) with long (blue or light gray) and short handles (red or dark gray) (color online). (B) Measured force variance for short (red or dark gray circles) and long handles (blue or light gray circles). (C) Measured effective stiffness, ɛeff (upper), stiffness of the trap, ɛb (middle), and stiffness of the molecular system, ɛx (lower) for short (red or dark gray circles) and long (blue or light gray circles) handles. Fits to the elastic model are shown for long (blue or light gray lines) and short (red or dark gray lines) handles. Results are the average over three and four different molecules for the short- and long-handle cases, respectively.

References

1. Ritort F. Single-molecule experiments in biological physics: methods and applications. J. Phys. Condens. Matter. 2006;18:531–583. - PubMed
1. Greenleaf W.J., Woodside M.T., Block S.M. Passive all-optical force clamp for high-resolution laser trapping. Phys. Rev. Lett. 2005;95:208102. - PMC - PubMed
1. Liphardt J., Onoa B., Bustamante C. Reversible unfolding of single RNA molecules by mechanical force. Science. 2001;292:733–737. - PubMed
1. Woodside M.T., Behnke-Parks W.M., Block S.M. Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins. Proc. Natl. Acad. Sci. USA. 2006;103:6190–6195. - PMC - PubMed
1. Woodside M.T., Anthony P.C., Block S.M. Direct measurement of the full, sequence-dependent folding landscape of a nucleic acid. Science. 2006;314:1001–1004. - PMC - PubMed

Improving signal/noise resolution in single-molecule experiments using molecular constructs with short handles - PubMed (original) (raw)