Abstract
Pseudouridine (Ψ) is the most common chemical modification present in RNA. In general, Ψ increases the thermodynamic stability of RNA. However, the degree of stabilization depends on the sequence and structural context. To explain experimentally observed sequence dependence of the effect of Ψ on the thermodynamic stability of RNA duplexes, we investigated the structure, dynamics and hydration of RNA duplexes with an internal Ψ-A base pair in different nearest-neighbor sequence contexts. The structures of two RNA duplexes containing 5′-GΨC/3′-CAG and 5′-CΨG/3′-GAC motifs were determined using NMR spectroscopy. To gain insight into the effect of Ψ on duplex dynamics and hydration, we performed molecular dynamics (MD) simulations of RNA duplexes with 5′-GΨC/3′-CAG, 5′-CΨG/3′-GAC, 5′-AΨU/3′-UAA and 5′-UΨA/3′-AAU motifs and their unmodified counterparts. Our results showed a subtle impact from Ψ modification on the structure and dynamics of the RNA duplexes studied. The MD simulations confirmed the change in hydration pattern when U is replaced with Ψ. Quantum chemical calculations showed that the replacement of U with Ψ affected the intrinsic stacking energies at the base pair steps depending on the sequence context. The calculated intrinsic stacking energies help to explain the experimentally observed sequence dependent changes in the duplex stability from Ψ modification.
Highlights
Pseudouridine (Ψ) is the most common chemical modification present in RNA
Structural studies of the P6.1 hairpin derived from the human telomerase RNA activation domain revealed that two Ψ modifications located in the loop increased both base-stacking and hydrogen-bonding interactions within the loop compared with the unmodified construct, resulting in higher thermodynamic stability
We previously reported the influence of pseudouridylation on the thermodynamic stability of RNA duplexes with Ψ modifications where the Ψ was located at various positions and formed base pairs with A, G, U and C31
Summary
Pseudouridine (Ψ) is the most common chemical modification present in RNA. In general, Ψ increases the thermodynamic stability of RNA. Structural studies of the P6.1 hairpin derived from the human telomerase RNA activation domain revealed that two Ψ modifications located in the loop increased both base-stacking and hydrogen-bonding interactions within the loop compared with the unmodified construct, resulting in higher thermodynamic stability. To further elucidate how the replacement of the middle U-A base pair with Ψ-A in different sequence contexts influences the structure, dynamics and hydration of RNA duplexes, we performed a systematic study using molecular dynamics (MD) simulations. We carried out MD simulations in explicit solvent for all duplexes with internal Ψ-A base pairs studied in previous work[31], namely, duplexes with 5′-GΨC/3′-CAG, 5′-CΨG/3′-GAC, 5′-AΨU/3′-UAA and 5′-UΨA/3′-AAU motifs and their unmodified counterparts. To understand how the replacement of U with Ψ changes the stacking interactions, we applied high-level ab initio methods and performed calculations for eight unique base pair steps for the RNA structures derived from MD simulations
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