Measuring the elasticity of ribonucleotide(s)-containing DNA molecules using AFM

Ribonucleotides, ribonucleoside monophosphates (rNMPs), have been revealed as possibly the most noncanonical nucleotides in genomic DNA. rNMPs, either not removed from Okazaki fragments during DNA replication or incorporated and scattered throughout the genome, pose a perturbation to the structure and a threat to the stability of DNA. The instability of DNA is mainly due to the extra 2'-hydroxyl (OH) group of rNMPs which give rise to local structural effects, which may disturb various molecular interactions in cells. As a result of these structural perturbations by rNMPs, the elastic properties of DNA are also affected. Here, we show the approach to test whether the presence of rNMPs in DNA duplexes could alter the elasticity of DNA by implementing atomic force microscopy (AFM)-based single molecule force-measurements of short rNMP(s)-containing oligonucleotides (oligos).