Pseudouridine residues as substrates for serum ribonucleases

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–O ^5″ bond on the 3′ side of pyrimidine residues. Pseudouridine (Ψ) is the most abundant modified nucleoside in natural RNA. The substitution of uridine (U) with Ψ or N ¹ -methylpseudouridine (m ¹ Ψ) 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–O ^5″ bond on the 3′ side of Ψ and m ¹ Ψ. We find that these enzymes catalyze the cleavage of UpA up to 10-fold more efficiently than the cleavage of ΨpA or m ¹ ΨpA. X-ray crystallography of enzyme-bound nucleoside 2′,3′-cyclic vanadate complexes and molecular dynamics simulations of enzyme·dinucleotide complexes show that U, Ψ, and m ¹ Ψ 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 p K _a of the 2′ hydroxy group of U and enhances its nucleophilicity toward the P–O ^5″ bond. Experimentally, we found that UpA does indeed undergo spontaneous hydrolysis faster than does m ¹ ΨpA. Our findings inform the continuing development of RNA-based vaccines and therapeutic agents.