The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R-loop sequences
June
The monoclonal S9.6 antibody exhibits highly variable binding affinities towards different R- loop sequences
Fabian KoÈ nig 0 1
Thomas Schubert 0 1
Gernot LaÈ ngst 0 1
0 Biochemistry III; Biochemistry Centre Regensburg (BCR), University of Regensburg, Universit aÈtsstr , Regensburg, Germany, 2 2Bind GmbH, Regensburg , Germany
1 Editor: Fenfei Leng, Florida International University , UNITED STATES
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 DNAÐRNA and also a high affinity (2.7 nM) for RNAÐRNA 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.
Data Availability Statement; All relevant data are within the paper
-
Funding: This work was funded by the DFG in the
framework of the SFB960. The 2bind GmbH,
Regensburg, Germany is a provider for biophysical
analytical services. 2bind contributed to this study
in data collection using MicroScale
Thermophoresis (MST).
Competing interests: Prof. Gernot LaÈngst is an
owner of 2bind and belongs to the scientific
advisory board. This does not alter our adherence
Introduction
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
]. The
persistence of R-loops can result in the accumulation of DNA double-strand breaks (DSBs)
[
4
], leading to DNA rearrangements and genome instability [
1,5
].
R-loops occur naturally during transcription and serve for example in class switch
recombination of immunoglobulin (Ig) genes in activated B cells [
6
] and are functional structures in
mitochondrial DNA replication [
7,8
]. Genome-wide mapping techniques were established to
determine R-loop occurrence in human, mouse, and yeast cells, revealing that R-loops are
highly abundant, with 5% of mammalian genomic sequences and 8% of the budding yeast
sequences forming R-loops [
9,10
]. Potential regulatory functions of these structures are
implied, as R-loop sequences are frequently identified at GC-rich regions such as many
promoters and 30end regions, where they appear to play significant roles in transcription [9,11±
13]. R-loops can now be effectively mapped with high-throughput methods that are based on
the specific recognition of RNA-DNA hybrids by the S9.6 antibody [
14,15
]. The antibody was
recently used to detect and localize DNAÐRNA hybrids that have been linked to genomic
instability, at CpG island promoters, terminator regions and genomic regions with altered
chromatin structure [16±19] [
9,20
].
The monoclonal antibody S9.6 was originally generated in mice using an in vitro
synthesized FX174 DNAÐRNA antigen and shown to exhibit high specificity and affinity for DNA
ÐRNA hybrids [
14
]. The antibody was initially used in assays to detect and quantify specific
RNA-DNA hybrids [21±23] and for genome wide array based hybridization mapping
techniques [
24,25
]. The specific recognition of miRNA-DNA hybrids with a length of 22nt was
also used to develop sensitive biosensor systems [
26,27
].
Because of the widespread use of the S9.6 antibodies in research and the importance to
interpret the specific binding events, a recent study sought to further characterize the binding
affinities and specificity of the single-chain variable fragment (scFv) of S9.6 [
15
]. Surface
Plasmon Resonance (SPR) experiments revealed a high binding affinity of 0.6 nM for DNA-RNA
hybrids and in addition an about 5 times lower and still high binding affinity for RNA-RNA
hybrids. The smallest epitope recognized by the antibody was shown to consist of 6 base pairs
[
15
]. In contrast, genome wide hybridisation mapping techniques suggest a minima (...truncated)