Koenig, Fabian and Schubert, Thomas and Laengst, Gernot (2017) The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different Rloop sequences. PLOS ONE, 12 (6): e0178875. ISSN 1932-6203,
Full text not available from this repository. (Request a copy)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 thermo-phoresis 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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | SOLUTION HYBRIDIZATION ASSAY; TRANSCRIPTIONAL PAUSE SITES; R-LOOP FORMATION; RIBOSOMAL-RNA; STRANDED-RNA; DNA-RNA; HYBRIDS; IMMUNODETECTION; INSTABILITY; TERMINATION; |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Biochemie III > Prof. Dr. Gernot Längst |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 14 Dec 2018 13:10 |
| Last Modified: | 26 Feb 2019 10:17 |
| URI: | https://pred.uni-regensburg.de/id/eprint/705 |
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