Pseudouridine residues as substrates for serum ribonucleases

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Clinical applications of RNA require stability in serum, which contains ribonucleases (RNases) like RNase 1.
• Pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ) are modified nucleosides that reduce mRNA immunogenicity and enhance translation, making them vital for RNA vaccines.

Purpose of the Study:

• To investigate the susceptibility of pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ) to cleavage by RNase A and RNase 1.
• To elucidate the molecular mechanisms underlying the differential cleavage rates of modified versus unmodified RNA.

Main Methods:

• Enzymatic assays comparing cleavage rates of UpA, ΨpA, and m1ΨpA by RNase A and RNase 1.
• X-ray crystallography of enzyme-bound nucleoside 2',3'-cyclic vanadate complexes.
• Molecular dynamics simulations of enzyme·dinucleotide complexes.
• Quantum chemistry calculations to assess intrinsic reactivity.

Main Results:

• RNase A and RNase 1 cleaved UpA up to 10-fold more efficiently than ΨpA or m1ΨpA.
• Structural analyses showed similar binding modes for U, Ψ, and m1Ψ with RNase A and RNase 1.
• Quantum chemistry and experimental hydrolysis data indicated that the increased reactivity of UpA is due to the intrinsic electronic properties of uridine.

Conclusions:

• The substitution of uridine with pseudouridine or N1-methylpseudouridine confers significant resistance to RNase-mediated cleavage.
• Understanding these stability differences is critical for optimizing the design and delivery of RNA-based therapeutics and vaccines.