The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The S9.6 monoclonal antibody is a key reagent for studying R-loop structures.
• Previous research indicated high affinity of S9.6 for DNA-RNA and RNA-RNA hybrids using surface plasmon resonance.

Purpose of the Study:

• To investigate the sequence specificity and binding affinities of the S9.6 antibody for various RNA-DNA hybrid sequences.
• To evaluate the impact of GC-content on S9.6 binding.
• To question the utility of S9.6 for quantitative R-loop analysis in vivo.

Main Methods:

• Microscale thermophoresis for surface-independent interaction studies.
• Electromobility shift assays to assess binding affinities.
• Evaluation of diverse RNA-DNA hybrid sequences with varying GC-content.

Main Results:

• S9.6 antibody exhibits high affinity for previously studied sequences.
• Binding affinities are highly sequence-specific, not solely dependent on GC-content.
• Identified R-loop sequences with no binding, micromolar, or nanomolar binding affinities.

Conclusions:

• The binding of the S9.6 antibody to R-loops is critically dependent on the specific RNA-DNA sequence.
• The S9.6 antibody's reliability for quantitative R-loop analysis in vivo is questionable due to sequence-specific binding.
• Further research is needed to develop more accurate methods for R-loop quantification.