Spin Labeling of Long RNAs Via Click Reaction and Enzymatic Ligation

Area of Science:

• Biochemistry
• Spectroscopy
• Molecular Biology

Background:

• Electron paramagnetic resonance (EPR) spectroscopy is crucial for studying biomacromolecular structure and dynamics.
• Site-specific labeling of oligonucleotides with spin labels is necessary for EPR applications.
• Existing methods may have limitations for labeling long oligonucleotide sequences.

Purpose of the Study:

• To develop and present a reliable protocol for site-specifically spin labeling long oligonucleotides.
• To enable structural and mechanistic investigations of complex RNA molecules using EPR.
• To provide a method for labeling riboswitches, ribozymes, and DNAzymes.

Main Methods:

• Spin labeling of short RNA strands with nitroxides using copper-(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or "click" reaction.
• Enzymatic ligation of the spin-labeled RNA strand to a longer RNA sequence to create the full-length construct.
• Application of the protocol to RNA sequences up to 81 nucleotides.

Main Results:

• The protocol demonstrates robustness and high yields for labeling RNA sequences up to 81 nucleotides.
• The method is adaptable for labeling longer RNA constructs, including riboswitches, ribozymes, and DNAzymes.
• Successful site-specific incorporation of nitroxide spin labels was achieved.

Conclusions:

• The developed protocol offers an effective strategy for spin labeling long oligonucleotides.
• This technique facilitates coarse-grained structure determination and mechanistic studies of RNA-based systems.
• The method expands the applicability of EPR spectroscopy to larger and more complex nucleic acid structures.