Azidopolynucleotides as photoaffinity reagents

Area of Science:

• Molecular Biology
• Biochemistry
• Biophysical Chemistry

Background:

• Understanding the subunit topography of RNA polymerase is crucial for elucidating transcription mechanisms.
• Photoaffinity labeling is a powerful technique for probing molecular interactions and structures in biological systems.

Purpose of the Study:

• To synthesize novel polynucleotides incorporating 8-azidoadenosine and 8-azidoinosine residues.
• To utilize these modified polynucleotides, specifically poly(adenylic, 8-azidoadenylic acid) [poly(A,z8A)], as photoaffinity reagents.
• To investigate the subunit topography of RNA polymerase from Escherichia coli using the synthesized photoaffinity reagent.

Main Methods:

• Enzymatic polymerization of nucleoside diphosphates using polynucleotide phosphorylase from Escherichia coli.
• Preparation of copolymers containing adenosine/8-azidoadenosine and inosine/8-azidoinosine.
• Complex formation between synthesized copolymers and complementary homopolymers (polyuridylic or polycytidylic acids).
• Application of single-stranded poly(A,z8A) as a photoaffinity reagent for RNA polymerase.

Main Results:

• Successfully synthesized polynucleotides containing 8-azidoadenosine and 8-azidoinosine residues.
• Demonstrated that these copolymers can form complexes with polyuridylic or polycytidylic acids.
• Poly(A,z8A) effectively served as a photoaffinity reagent to probe the subunit topography of E. coli RNA polymerase.

Conclusions:

• Novel azido-modified polynucleotides can be synthesized enzymatically.
• These modified polynucleotides are suitable for forming complexes and can function as photoaffinity labels.
• The study provides a new tool for investigating the structural organization of RNA polymerase.