Analysis of RNA flexibility by scanning force spectroscopy

Abstract

Scanning force spectroscopy was used to measure the mechanical properties of double stranded RNA molecules in comparison with DNA. We find that, similar to the B-S transition in DNA, RNA molecules are stretched from the assumed A' conformation to a stretched conformation by applying a defined force (plateau force). The force depends on the G + C content of the RNA and is distinct from that required for the B-S transition of a homologous DNA molecule. After the conformational change, DNA can be further extended by a factor of 0.7 +/- 0.2 (S-factor) before melting occurs and the binding of the molecule to the cantilever is finally disrupted. For RNA, the S-factor was higher (1.0 +/- 0.2) and more variable. Experiments to measure secondary structures in single stranded RNA yielded a large number of different force-distance curves, suggesting disruption and stretching of various secondary structures. Oriented attachment of the molecules to the substrate, a defined pick-up point and an increased resolution of the instrument could provide the means to analyse RNA secondary structures by scanning force spectroscopy.

Double-stranded RNA under force and torque: Similarities to and striking differences from double-stranded DNA

“…S2B) and an overstretching transition for torsionally unconstrained molecules (SI Appendix, Fig. S2C), in agreement with previous single-molecule studies (16,17). From fits of the extensible WLC model, we found S RNA = 350 ± 100 pN, about threefold lower than S DNA (SI Appendix, Fig.…”

“…1A). Although recent single-molecule stretching experiments using torsionally unconstrained dsRNA have revealed its bending persistence length (15,16), stretch modulus (16), and an overstretching transition (16,17), its response to torsional strains and structural transitions under forces and torques is unknown. This dearth of information on dsRNA is partially due to the relative difficulty, compared with dsDNA, of assembling RNA constructs suitable for single-molecule force and torque measurements.…”

Mesoscopic model parametrization of hydrogen bonds and stacking interactions of RNA from melting temperatures

“…Early studies indicated a large persistence length of RNA when compared with DNA (1), that is that RNA is more rigid than DNA. This is still largely the view which was supported by single molecule (2,3) and atomic force microscopy (AFM) measurements (4). On a base pair level, that is, on a microscopic scale, most of our knowledge comes from molecular dynamic simulations (5–8).…”

Atomic Force Microscopy Analysis of Icosahedral Virus RNA

“…The forces were similar to those employed in earlier studies on viruses 46,53,54 and biological crystals. [55][56][57] There is considerable previous AFM work concerning double-stranded DNA 49,58,59 but, with some exceptions, 58,[60][61][62] rather little on single-stranded RNA. Before investigating single-stranded RNA, DNA plasmids pET19B (Sigma Biochemical Co. St. Louis, MO) were spread on mica and imaged.…”