GC-content dependence of elastic and overstretching properties of DNA:RNA hybrid duplexes

Abstract DNA:RNA hybrid duplex plays important roles in various biological processes. Both its structural-stability and interactions with proteins are highly sequence-dependent. In this study, we utilize homebuilt optical tweezers to investigate how GC-contents in the sequence influence the structural and mechanical properties of DNA:RNA hybrid by measuring its contour length, elasticities and over-stretching dynamics. Our results support that DNA:RNA hybrid adopts a conformation between the A- and B-form helix and the GC-content does not affect its structural and elastic parameters obviously when varying from 40% to 60% before the overstretching transition of DNA:RNA hybrid occurs. In the overstretching transition, however, our study unravels significant heterogeneity and strong sequence dependence, suggesting the presence of a highly dynamic competition between the two processes, namely the S-form duplex formation (non-hysteretic) and the unpeeling (hysteretic). Analyzing the components left in DNA:RNA hybrid after the overstretching transition suggests that the RNA strand is more easily unpeeled than the DNA strand while an increase in the GC-content from 40% to 60% can significantly reduce unpeeling. Large hysteresis is observed between the stretching and relaxation processes, which is also quantitatively correlated with the percentage of unpeeling in DNA:RNA duplex. Increasing in both the salt concentration and GC-content can effectively reduce the hysteresis with the latter being more significant. Together, our study reveals that the mechanical properties of DNA:RNA hybrid duplexes are significantly different from dsDNA and dsRNA and its over-stretching behavior is highly sequence-dependent. These results should be taken into account in the future studies on DNA:RNA hybrid-related functional structures and motor proteins.