Thermodynamic characterization and nearest neighbor parameters for RNA duplexes under molecular crowding conditions

Abstract

It is essential to study RNA under molecular crowding conditions to better predict secondary structures of RNAs in vivo . No systematic study has been completed to determine the effects of molecular crowding on RNA duplexes of varying lengths and sequence composition. Here, optical melting, circular dichroism, and osmometry data were collected for RNA duplexes in a 20% polyethylene glycol (with an average molecular weight of 200 g/mol) solution (PEG 200), and nearest neighbor parameters were derived using this data. RNA duplexes are destabilized, on average, 1.02 kcal/mol in the presence of 20% PEG 200. The ΔG° 37 values predicted by the nearest neighbor parameters for RNA duplexes in 20% PEG 200 were ∼0.65 kcal/mol closer to experimental Δ G ° 37 values than those predicted by the standard nearest neighbor model. For one DNA sequence in solution with small crowders, the Δ G ° 37 values predicted by the 20% PEG 200 RNA nearest neighbor parameters were closer to the experimental values than Δ G ° 37 values predicted by either the RNA or DNA standard nearest neighbor models. This indicates that the nearest neighbor parameters for RNA duplexes in 20% PEG 200 may be generalizable to RNA and DNA duplexes in solutions with small crowding agents.

Nearest-neighbor parameters for predicting DNA duplex stability in diverse molecular crowding conditions

“…Among the 36 DNA tested duplexes (SI Appendix, Table S4), the average differences in ΔH°, ΔS°, and ΔG°3 7 between the predicted and measured values were 5.7, 6.3, and 4.3%, respectively, which were within the limits of reported NN parameters (9)(10)(11)18). The average difference for T m was also very small (1.0°C) compared with those for DNA (2.0°C) (11) and RNA (1.9°C) (9) duplexes, using the NN model in the absence of cosolute.…”

“…However, since classical NN parameters are determined under conditions of 1 M NaCl, it is difficult to apply them for predictions in intracellular environments populated with highly concentrated macromolecules in lower salt concentrations. Recent attempts to establish NN parameters applicable to physiological conditions have aimed to predict the intracellular stability of duplexes (13)(14)(15)(16)(17)(18)(19). Although the ΔG°3 7 of DNA and RNA duplexes under modified conditions was predicted, such predictions can only be used in a specific condition.…”

Dielectricity of a molecularly crowded solution accelerates NTP misincorporation during RNA-dependent RNA polymerization by T7 RNA polymerase

“…A high cosolute concentration is associated with molecular crowding, which critically changes the stability of nucleic acids due to decreases in the dielectric constant 8 , decreases in water activity 9 – 11 , increases in viscosity 12 , and increases in excluded volume 13 . In particular, the Watson–Crick base pairs become unstable, although the edge of the duplex is stabilized under molecular crowding conditions 14 – 16 . Furthermore, molecular crowding only slightly destabilizes duplex structures having mismatched base pairs compared to that without mismatched pairs 17 .…”

Atomistic Simulation of Lysozyme in Solutions Crowded by Tetraethylene Glycol: Force Field Dependence

“…On the contrary, soft interactions with PEG were also observed to induce structural changes and destabilize proteins [ 28 , 31 , 32 , 33 , 34 ]. For the nucleic acids, low molecular weight PEGs were observed to decrease their thermodynamic stability, while high molecular weight PEGs showed the opposite [ 35 ]. PEG-induced destabilization of biomolecules is likely due to the stronger “soft” interactions relative to the “hard” interactions of excluded volume; the former offers either favorable or unfavorable contributions, while the latter leads to compacted structures and is usually thermodynamically favorable [ 12 ].…”