Real time measurements of elongation by a reverse transcriptase using surface plasmon resonance

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Understanding polymerase processivity and drug inhibition mechanisms is crucial.
• Existing assays for monitoring polymerization can be indirect or lack real-time kinetic data.

Purpose of the Study:

• To develop a rapid, direct assay for measuring polymerase-induced DNA/RNA elongation.
• To utilize surface plasmon resonance (SPR) for real-time kinetic analysis of polymerization.
• To assess the impact of chain-terminating drugs on polymerase activity.

Main Methods:

• Immobilization of DNA.RNA and DNA.DNA hybrids on a biotin-streptavidin surface.
• Monitoring real-time changes in mass via SPR signal during nucleotide addition.
• Utilizing a chain terminator drug (3'-deoxy-3'-azidothymidine triphosphate) to validate the assay's sensitivity to elongation inhibition.

Main Results:

• The SPR assay successfully detected increases in mass corresponding to primer strand elongation.
• Polymerization extent and rate were estimated in real-time.
• 3'-deoxy-3'-azidothymidine triphosphate completely inhibited the signal increase, confirming assay specificity for elongation inhibition.

Conclusions:

• Surface plasmon resonance provides a sensitive and direct method for assaying polymerase elongation kinetics.
• This technique facilitates the study of polymerase-template affinities and the efficacy of elongation inhibitors.
• The developed assay is valuable for drug discovery and fundamental research on nucleic acid polymerization.