Mechanical characterization of base analogue modified nucleic acids by force spectroscopy

Abstract

We use mechanical unfolding of single DNA hairpins with modified bases to accurately assess intra- and intermolecular forces in nucleic acids. As expected, the modification stabilizes the hybridized hairpin, but...

Exploring the conformational dynamics of the SARS-CoV-2 SL4 hairpin by combining optical tweezers and base analogues

“…In a recent study, we showed that the tC BA can stabilize a fully complementary DNA hairpin. 35 Here we demonstrate how BAs can be used in optical tweezers experiments to explore conformational states of RNA structures and transitions between them. Since BAs are similar in size to natural bases and typically designed to (1) maintain specificity for complementary bases; (2) restore hydrogen bonds with their complements; and/or (3) preserve the intrinsic secondary structure of nucleic acids, their incorporation into an RNA/DNA of interest enables studies of a selected region within the molecule with base-pair resolution.…”

“…In our previous optical tweezers study using the tC BA in a fully complementary DNA hairpin, we observed an increase in the hairpin's mechanical stability with tC incorporation. 35 tC is chemically similar to tC O (ref. 34 ) and in UV melting studies, both tC O (ref.…”

“…Short handles are advantageous for data analysis, since as an effect of using them the force changes approximately linearly with extension and the signal-to-noise ratio is improved. In comparison to the short DNA handles presented so far in a few DNA folding studies, 35,50 our protocol has the advantage that it does not involve ligation in the studied nucleic acid sequence, which is particularly important for stems containing secondary-structure elements, which are common in, for example, pre-miRNAs and viral RNA hairpins. In addition, it involves a purification step (Fig.…”

“…27,32,33 In a previous study, we used tC (differing in structure to tC O only in that the oxygen in the middle ring is replaced by a sulfur) 34 in place of cytosine in a fully complementary 44 ntlong DNA hairpin and observed an increase in its stability in unfolding experiments with optical tweezers with the number of tC incorporations. 35 The added value of using BAs compared to standard mutagenesis, where an intrinsic nucleobase is replaced by another canonical base 15,21,22,36 or where it is omitted, 16 is that more subtle, close-to-native effects can be observed and that specific intramolecular interactions can be investigated.…”

Complex Conformational Dynamics of the Heart Failure-Associated Pre-miRNA-377 Hairpin Revealed by Single-Molecule Optical Tweezers

“…These dsDNAs served as handles to attach the molecule to beads coated with streptavidin and anti-digoxigenin, respectively (Figure 1B). To the best of our knowledge, short DNA handles have never been used before in optical tweezers studies of RNA molecules, but merely in a few DNA-folding studies [25][26][27][28]. The advantage of using short DNA handles over the more common long handles (typically~500 bp-a few kbp) [18,[29][30][31][32][33] is that they improve signal-to-noise and make the force changes approximately linear with extension (Figure 1C), which significantly benefits the data analysis [25].…”