High-throughput thermal stability assessment of DNA hairpins based on high resolution melting.

On the basis of high-resolution melting, a high-throughput approach to measure melting temperatures (Ts) of short DNA hairpins was developed. With this method, Ts of thousands of triloop, tetraloop, and pentaloop hairpins involving various loop sequences and various closing base pairs (cbp) were obtained in hours. The stability of triloop hairpins decreased with the change of cbp (5'-3') in the order of c-g > g-c > t-a ≥ a-t, showing that the cbp of 5'-Pyr-Pur-3' (Pyr = pyrimidine, Pur = purine) contributed more stability than 5'-Pur-Pyr-3'. For tetraloop hairpins, GNNA, GNAB, and CNNG (N = A, G, C, or T; B = G, C, or T) were found to be highly stable irrespective of the cbp type. TNNA was also stable in both g-c and a-t families, while CGNA only in the c-g family. Pentaloop hairpins of cTGNAGg, cGNYNAg (Y = T or C) and cCGNNAg were exceptionally stable motifs. In most cases, pyrimidine-rich loops were more favorable to stabilize the whole structure than purine-rich ones. The present approach showed a good performance in assessing the thermal stability of large amounts of DNA hairpins comprehensively. These data are useful to understand the sequence dependence of the stability of DNA secondary structures and promising to improve the structure simulation by consummating basic databases.