Pseudouridine residues as substrates for serum ribonucleases

Abstract

In clinical uses, RNA must maintain its integrity in serum that contains ribonucleases (RNases), especially RNase 1, which is a human homolog of RNase A. These omnipresent enzymes catalyze the cleavage of the P-O5'' bond on the 3' side of pyrimidine residues. Pseudouridine ({Psi}) is the most abundant modified nucleoside in natural RNA. The substitution of uridine (U) with {Psi} or N1-methylpseudouridine (m1{Psi}) reduces the immunogenicity of mRNA and increases ribosomal translation, and these modified nucleosides are key components of RNA-based vaccines. Here, we assessed the ability of RNase A and RNase 1 to catalyze the cleavage of the P-O5'' bond on the 3' side of {Psi} and m1{Psi}. We find that these enzymes catalyze the cleavage of UpA up to 10-fold more efficiently than the cleavage of {Psi}pA or m1{Psi}pA. X-ray crystallography of enzyme-bound nucleoside 2',3'-cyclic vanadate complexes and molecular dynamics simulations of enzyme{middle dot}dinucleotide complexes show that U, {Psi}, and m1{Psi} bind to RNase A and RNase 1 in a similar manner. Quantum chemistry calculations suggested that the higher reactivity of UpA is intrinsic, arising from an inductive effect that decreases the pKa of the 2'-hydroxy group of U and enhances its nucleophilicity toward the P-O5'' bond. Experimentally, we found that UpA does indeed undergo spontaneous hydrolysis faster than does m1{Psi}pA. Our findings inform the continuing development of RNA-based vaccines and therapeutic agents.