Abstract
The monoclonal antibody S9.6 is a widely-used tool to purify, analyse and quantify R-loop structures in cells. A previous study using the surface plasmon resonance technology and a single-chain variable fragment (scFv) of S9.6 showed high affinity (0.6 nM) for DNA—RNA and also a high affinity (2.7 nM) for RNA—RNA hybrids. We used the microscale thermophoresis method allowing surface independent interaction studies and electromobility shift assays to evaluate additional RNA-DNA hybrid sequences and to quantify the binding affinities of the S9.6 antibody with respect to distinct sequences and their GC-content. Our results confirm high affinity binding to previously analysed sequences, but reveals that binding affinities are highly sequence specific. Our study presents R-loop sequences that independent of GC-content and in different sequence variations exhibit either no binding, binding affinities in the micromolar range and as well high affinity binding in the nanomolar range. Our study questions the usefulness of the S9.6 antibody in the quantitative analysis of R-loop sequences in vivo.
Highlights
R-loops are local RNA-DNA hybrid sequences, generally formed by a nascent G-rich transcript hybridizing with the DNA template strand and thereby leaving the non-template DNA single stranded [1]
Oligonucleotides were used in microscale thermophoresis (MST) and electromobility shift assays (EMSA) at concentrations ranging from 1 nM to 40 nM, depending on the binding affinity and Nanotemper device used for MST analysis
To test the sequence specificity of S9.6 antibody binding to R-loops, we designed a set of oligonucleotides, with varying GC-content and length (Table 1)
Summary
R-loops are local RNA-DNA hybrid sequences, generally formed by a nascent G-rich transcript hybridizing with the DNA template strand and thereby leaving the non-template DNA single stranded [1]. These structures were first described in vitro in 1976 and about 20 years ago in prokaryotes having a mutation in the Topoisomerase I gene [2]. R-loops were initially considered as a by-product of transcription, but during the past decade very important functions of R-loops in transcription, genomic stability and a variety of diseases emerged [3].
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