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Locked nucleic acid oligomers as handles for single molecule manipulation.

Berezney JP, Saleh OA - Nucleic Acids Res. (2014)

Bottom Line: A simple, minimally-perturbative labeling strategy would significantly broaden the possible applications of SMM experiments, perhaps even allowing the study of native biomolecular structures.Our results show that LNA provides a simple, stable means to functionalize dsDNA for manipulation.We provide design rules that will facilitate their use in future experiments.

View Article: PubMed Central - PubMed

Affiliation: Materials Department, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.

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(A) LNA-1 oligomers bind specifically to the target position (1453 nm, 4404 bp) within the full 48.5 kilobases of the lambda phage genome, N = 17, bin = 500 nm (light gray bars). LNA oligomers also bind specifically when the lambda genome orientation is flipped (dark gray bars), N = 24. (B) LNA-2 oligomers bind specifically to the target position (39 138 bp, 3183 nm) within the lambda phage genome, N = 30, bin = 500 nm. (C) LNA-3 oligomers bind specifically to the target position (4525 bp, 1352 nm) within the pMAL-p5x vector, N = 24, bin = 200 nm. Error bars represent +/− 1 standard deviation.
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Figure 2: (A) LNA-1 oligomers bind specifically to the target position (1453 nm, 4404 bp) within the full 48.5 kilobases of the lambda phage genome, N = 17, bin = 500 nm (light gray bars). LNA oligomers also bind specifically when the lambda genome orientation is flipped (dark gray bars), N = 24. (B) LNA-2 oligomers bind specifically to the target position (39 138 bp, 3183 nm) within the lambda phage genome, N = 30, bin = 500 nm. (C) LNA-3 oligomers bind specifically to the target position (4525 bp, 1352 nm) within the pMAL-p5x vector, N = 24, bin = 200 nm. Error bars represent +/− 1 standard deviation.

Mentions: Overview of strategy to assay LNA/DNA binding strength and specificity in a magnetic tweezer: (A) Biotin-labeled LNA oligomers bind to surface-bound DNA, and mediate the tethering of a 1 μm streptavidin-coated magnetic bead. The tether is extended by a known force through application of a magnetic field, and its length is measured by optical bead tracking. (B) Force/length relations for individual tethers are fit to theory; one representative fit is plotted in black. The fits return estimates of each tether's persistence length, lp, and contour length, Lc; the latter is an estimate of the LNA/DNA binding position. Multiplexed measurements permit elastic measurements of many tethers. Histograms of the fit contour lengths report on the characteristics of the NAA/DNA interaction. The 17 force/extension curves shown here correspond to the histogram (gray bars) in Figure 2A.


Locked nucleic acid oligomers as handles for single molecule manipulation.

Berezney JP, Saleh OA - Nucleic Acids Res. (2014)

(A) LNA-1 oligomers bind specifically to the target position (1453 nm, 4404 bp) within the full 48.5 kilobases of the lambda phage genome, N = 17, bin = 500 nm (light gray bars). LNA oligomers also bind specifically when the lambda genome orientation is flipped (dark gray bars), N = 24. (B) LNA-2 oligomers bind specifically to the target position (39 138 bp, 3183 nm) within the lambda phage genome, N = 30, bin = 500 nm. (C) LNA-3 oligomers bind specifically to the target position (4525 bp, 1352 nm) within the pMAL-p5x vector, N = 24, bin = 200 nm. Error bars represent +/− 1 standard deviation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231729&req=5

Figure 2: (A) LNA-1 oligomers bind specifically to the target position (1453 nm, 4404 bp) within the full 48.5 kilobases of the lambda phage genome, N = 17, bin = 500 nm (light gray bars). LNA oligomers also bind specifically when the lambda genome orientation is flipped (dark gray bars), N = 24. (B) LNA-2 oligomers bind specifically to the target position (39 138 bp, 3183 nm) within the lambda phage genome, N = 30, bin = 500 nm. (C) LNA-3 oligomers bind specifically to the target position (4525 bp, 1352 nm) within the pMAL-p5x vector, N = 24, bin = 200 nm. Error bars represent +/− 1 standard deviation.
Mentions: Overview of strategy to assay LNA/DNA binding strength and specificity in a magnetic tweezer: (A) Biotin-labeled LNA oligomers bind to surface-bound DNA, and mediate the tethering of a 1 μm streptavidin-coated magnetic bead. The tether is extended by a known force through application of a magnetic field, and its length is measured by optical bead tracking. (B) Force/length relations for individual tethers are fit to theory; one representative fit is plotted in black. The fits return estimates of each tether's persistence length, lp, and contour length, Lc; the latter is an estimate of the LNA/DNA binding position. Multiplexed measurements permit elastic measurements of many tethers. Histograms of the fit contour lengths report on the characteristics of the NAA/DNA interaction. The 17 force/extension curves shown here correspond to the histogram (gray bars) in Figure 2A.

Bottom Line: A simple, minimally-perturbative labeling strategy would significantly broaden the possible applications of SMM experiments, perhaps even allowing the study of native biomolecular structures.Our results show that LNA provides a simple, stable means to functionalize dsDNA for manipulation.We provide design rules that will facilitate their use in future experiments.

View Article: PubMed Central - PubMed

Affiliation: Materials Department, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.

Show MeSH
Related in: MedlinePlus