Abstract 2226 Elucidation of the binding characteristics of the DNA-binding cytokine, High Mobility Group B1 (HMGB1) toward single-stranded DNA

The majority of HMB1 related DNA studies focus on bent/distorted double stranded DNA substrates. Seminal studies by Yanai et al provided initial evidence that HMGB1 also binds to single stranded DNA (ss-DNA). Their work shows HMGB1 binds immunogenic ss-DNA and works in concert with cytosolic Toll-like Receptors (TLRs) to boost the innate immune response. In this capacity, HMGB1 functions as a "nucleic acid sensor." Subsequent studies show that non-immunogenic ss-DNA also bind HMGB1. Non-immunogenic ss-DNA offer an ideal method to target HMGB1 because these nucleic acids sequester HMGB1. Hence, non-immunogenic ss-DNA were explored further as ligands directed against HMGB1. This strategy is important because HMGB1 is a highly abundant (106 copies/cells) DNA-binding cytokine. Moreover, HMGB1 dysfunction is associated with a plethora of diseases/conditions that range from: lupus, rheumatoid arthritis, sepsis etc. Research shows that ss-DNA are viable ligands for HMGB1 but the binding mechanism is unknown. Our objective is to use biochemical wet lab and computational assays to further elucidate HMGB1 binding mechanisms toward ss-DNA to improve the efficacy of these substrates. To probe the features of HMGB1 binding toward ss-DNA, several biochemical techniques are used including: nuclease cleavage assays, electrophoretic mobility shift assays, fluorescence thermal shift (FTS) assays, circular dichroism (CD) and computational assays. The nuclease cleavage assays show, as expected, ss-DNA composed of phosphorothioate bonds (PS) are more resistant than natural ss-DNA substrates. PS containing ss-DNA are inert vs. three endo- and exonucleases (DNase I, Exonuclease III and T5 Exonuclease). Surprisingly, EMSAs indicate that HMGB1 does not bind preferentially toward PS ss-DNA – these data are not consistent chromatin pull-down assays. FTS studies show that HMGB1 binds preferentially toward PS ss-DNA. CD and computational studies are used to probe the structure of ss-DNA (PS and natural) to determine if the secondary structure of these substrates possess unique motifs that promote HMGB1 recognition. Preliminary CD studies do not provide data that indicates ss-DNA possess structural features that promote/enhance HMGB1 binding. However, computational studies show that PS ss-DNA possesses distinct structural conformers that promote/enhance HMGB1 recognition. Docking studies are ongoing to determine if small molecules can reveal potential structural motifs within model PS ss-DNA that promote HMGB1 binding. Taken collectively, our data shows that PS containing ss-DNA are viable HMGB1 ligands that should be investigated further.