Rolling circle amplification shows a sinusoidal template length-dependent amplification bias

Abstract

Biophysical properties of DNA such as its longitudinal and torsional persistence length govern many processes and phenomena in biology, DNA nanotechnology and biotechnology. It has, for example, long been known that the circularization efficiency of short DNA fragments shows a periodic pattern where fragments with integer helical turns circularize much more efficiently than those with odd helical half turns due to stronger stacking of duplex ends. Small DNA circles can serve as templates for rolling circle amplification (RCA), which is a common and extremely robust amplification mechanism for nucleic acids. We discovered a strong template length-dependent amplification efficiency bias of RCA with the same periodicity as B-DNA. However, stacking cannot explain the mechanism behind this bias as the presence of the polymerase in the bifurcation fork inhibits base stacking of ends. Instead, coarse-grained molecular dynamics simulations imply that different amplification efficiencies come from a varying fraying probability of the last two downstream base pairs. We conclude that an increased strain-promoted fraying probability can increase the polymerization rate compared to a relaxed template.

Determinants of cyclization–decyclization kinetics of short DNA with sticky ends

“…Although understanding energetics of DNA looping at even shorter lengths (<100 bp) is of growing interest, we argue that the J factor is neither a theoretically relevant nor an experimentally accessible quantity in this regime. As DNA becomes shorter, end segments are more flexible than internal segments [54], and end base pairs are more prone to fraying [47,55,56]. Moreover, discreteness of base pairs and sequence-dependent effects cannot be sufficiently averaged out over several helical turns.…”

A multiplex platform for digital measurement of circular DNA reaction products

“…Joffroy and coworkers recently reported that the efficiency of RCA depends on the size of the DNA circle templating the reaction. The efficiency was shown to vary in a periodic fashion with a maximum every 10.5 nucleotide, a number that corresponds to the length of one turn of the DNA double helix ( 13 ). We were able to confirm these results, as our results clearly demonstrate that circular templates of 68 and 79 nucleotides (previously described maxima) generated considerably brighter RCPs compared to ones arising from circular DNA templates of 63 and 73 nucleotides (previously described minima) (Figure 2B and Supplementary Figure S1B ).…”

“…Polyethylene glycol (PEG) has previously been shown to influence the efficiency of RCA on magnetic beads ( 12 ). Also the size and sequence composition of the circular templates have been reported to affect the efficiency of RCA performed in solution ( 13 , 14 ). Here we have optimized RCA performed on streptavidin-coated microscope slides.…”

Measuring Internal Forces in Single-Stranded DNA

“…In addition to its utility in computing forces in ssDNAbased force sensors, force clamp devices, and tensegrity nanostructures, our method can be expanded to calculate forces in more complex structures, such as DNA minicircles. 84,85 Furthermore, the method is general enough to be adapted to calculate forces in alternative coarse-grained models, including for non-DNA polymers like proteins.…”