The tiniest epitope identified by the antibody was shown to consist of 6 base pairs [15]. the S9.6 antibody in the quantitative analysis of R-loop sequences in 1976 and about 20 years ago in prokaryotes possessing a mutation in the Topoisomerase I gene [2]. R-loops were in the beginning 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 build up of DNA double-strand breaks (DSBs) [4], leading PETCM to DNA rearrangements and genome instability [1,5]. R-loops happen naturally during transcription and serve for example in class switch recombination of immunoglobulin (Ig) genes in triggered B cells [6] and are functional constructions in mitochondrial DNA replication [7,8]. Genome-wide mapping techniques were founded to determine R-loop event in human being, mouse, and candida cells, exposing that R-loops are highly abundant, with 5% of mammalian genomic sequences and 8% of the budding candida sequences forming R-loops [9,10]. Potential regulatory functions of these constructions are implied, as R-loop sequences are frequently recognized at GC-rich areas such as many promoters and 3end areas, where they appear to play significant functions in transcription [9,11C13]. R-loops can now be efficiently mapped with high-throughput methods that are based on the specific acknowledgement of RNA-DNA hybrids from the S9.6 antibody [14,15]. The antibody was recently used to detect and localize DNARNA hybrids that have been linked to genomic instability, at CpG island promoters, terminator areas and genomic areas with modified chromatin structure [16C19] [9,20]. The monoclonal IFNW1 antibody S9.6 was originally generated in mice using an synthesized X174 DNARNA antigen and shown to show high specificity and affinity for DNARNA hybrids [14]. The antibody was initially used in assays to detect and quantify specific RNA-DNA hybrids [21C23] and for genome wide array centered hybridization mapping techniques [24,25]. The specific acknowledgement of miRNA-DNA hybrids having a length of 22nt was also used to develop sensitive biosensor systems [26,27]. Because of the widespread use of the S9.6 antibodies PETCM 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 occasions lower and still high binding affinity for RNA-RNA hybrids. The smallest epitope identified by the antibody was shown to consist of 6 foundation pairs [15]. In contrast, genome wide hybridisation mapping techniques suggest a minimal binding length of about 15 bp, PETCM which exhibits half of the binding affinity when compared to 60 bp long RNA-DNA hybrids [25]. Since RNA-RNA duplexes form an A-helix structure that deviates from your RNA-DNA duplex structure [28], we suggest that the S9.6 antibody does not recognize the R-loop structure independent of R-loop sequence. To test this hypothesis, we used microscale thermophoresis (MST) and electromobility shift assays (EMSA) as with solution methods, in contrast to SPR, to determine binding affinities. Indeed, our results do suggest that the binding affinity of the S9.6 antibody varies with R-loop sequences, independent of the GC-content, exposing many sequence variants with no, or low binding affinities. Materials and methods Synthesis of nucleic acid hybrids DNA and RNA oligonuclotides were synthesized by Sigma-Aldrich (Germany) and cross RNA-DNA oligonucleotides were synthesized by Integrated DNA Systems (Coralville, IA, USA). All hybrids were synthesized with 5 Cy3, Cy5 or FAM fluorescence labels. To prepare RNA-DNA hybrids, the oligonucleotides were combined in equimolar ratios in Annealing Buffer (80 mM NaCl; 10 mMTris, pH 7.6, 1.5 mM MgCl2) heated to 95C for 3 minutes and then slowly cooled down (10 min) to room temperature. Oligonucleotides were used in microscale thermophoresis (MST) and electromobility shift assays (EMSA) at concentrations ranging PETCM from 1 nM to 40 nM, depending on the binding affinity and Nanotemper device utilized for MST analysis. Microscale thermophoresis MST experiments.