RNAs synthesized using photocleavable biotinylated nucleotides have dramatically improved catalytic efficiency

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Therapeutics

Background:

• Obtaining homogeneous, natively folded RNA populations is critical for structural and enzymatic studies.
• Traditional RNA preparation methods often involve denaturing procedures, compromising RNA integrity and activity.
• This limitation hinders detailed investigation and therapeutic applications of complex RNA molecules.

Purpose of the Study:

• To develop a facile, non-denaturing strategy for preparing homogeneous, catalytically active RNA populations.
• To demonstrate the utility of a novel biotin-PC GMP reagent for RNA synthesis.
• To compare the catalytic activity of RNAs prepared via native versus denaturing methods.

Main Methods:

• Utilized 'click' chemistry to couple biotin to a photocleavable (PC) guanosine monophosphate (GMP).
• Employed T7 RNA polymerase to transcribe RNAs using the modified biotin-PC GMP, yielding RNAs from 27 to 527 nucleotides.
• Assessed catalytic activity of minimal group II intron ribozymes using an in-gel fluorescence assay.

Main Results:

• Successfully synthesized biotin-PC GMP with high yield, which was accepted by T7 RNA polymerase.
• Transcribed a range of RNA molecules (27-527 nt) using the developed method.
• Natively prepared 160 and 175 kDa minimal group II intron ribozymes exhibited enhanced catalytic activity compared to refolded, denatured counterparts.

Conclusions:

• Non-denaturing RNA preparation yields homogeneous populations with superior catalytic activity.
• The developed click chemistry approach provides a simple and effective strategy for native RNA synthesis.
• This method holds significant promise for advancing RNA biophysics, structural biology, and therapeutic RNA development.