Thermodynamic contribution and nearest-neighbor parameters of pseudouridine-adenosine base pairs in oligoribonucleotides

Abstract

Pseudouridine (Ψ) is the most common noncanonical nucleotide present in naturally occurring RNA and serves a variety of roles in the cell, typically appearing where structural stability is crucial to function. Ψ residues are isomerized from native uridine residues by a class of highly conserved enzymes known as pseudouridine synthases. In order to quantify the thermodynamic impact of pseudouridylation on U-A base pairs, 24 oligoribonucleotides, 16 internal and eight terminal Ψ-A oligoribonucleotides, were thermodynamically characterized via optical melting experiments. The thermodynamic parameters derived from two-state fits were used to generate linearly independent parameters for use in secondary structure prediction algorithms using the nearest-neighbor model. On average, internally pseudouridylated duplexes were 1.7 kcal/mol more stable than their U-A counterparts, and terminally pseudouridylated duplexes were 1.0 kcal/mol more stable than their U-A equivalents. Due to the fact that Ψ-A pairs maintain the same Watson-Crick hydrogen bonding capabilities as the parent U-A pair in A-form RNA, the difference in stability due to pseudouridylation was attributed to two possible sources: the novel hydrogen bonding capabilities of the newly relocated imino group as well as the novel stacking interactions afforded by the electronic configuration of the Ψ residue. The newly derived nearest-neighbor parameters for Ψ-A base pairs may be used in conjunction with other nearest-neighbor parameters for accurately predicting the most likely secondary structure of A-form RNA containing Ψ-A base pairs.

Keywords: RNA thermodynamics; modified nucleotides; nonstandard nucleotides; secondary structure prediction.

FIGURE 1.

Pseudouridine (Ψ) synthases isomerize uridine residues, resulting in the creation of a new hydrogen bond donor (N1H) in the major groove of the Ψ-A pair (indicated by arrow).

FIGURE 2.

Electrostatic maps for Ψ and uridine using a methyl group to simulate the electronic contribution of the ribose ring. These maps were generated in Gaussian 09 (Frisch et al. 2009) using previously computed geometries for uridine (Johnson et al. 2011) with the corresponding atoms being substituted to produce Ψ.

FIGURE 3.

The ability of the relocated imino group (purple) on Ψ (green) to impact stacking interactions is likely decided by the positioning (5′ or 3′) of Ψ relative to the adjacent base (orange). Stacks shown are as follows: (A) 5′-UΨ; (B) 5′-GΨ; (C) 5′-ΨU; and (D) 5′-ΨG. These stacks were generated using InsightII (Accelrys) for geometries and PyMOL (Schrodinger 2010) for visualization.