Detection of viral RNAs at ambient temperature via reporter proteins produced through the target-splinted ligation of DNA probes.

Nucleic acid assays are not typically deployable in point-of-care settings because they require costly and sophisticated equipment for the control of the reaction temperature and for the detection of the signal. Here we report an instrument-free assay for the accurate and multiplexed detection of nucleic acids at ambient temperature. The assay, which we named INSPECTR (for internal splint-pairing expression-cassette translation reaction), leverages the target-specific splinted ligation of DNA probes to generate expression cassettes that can be flexibly designed for the cell-free synthesis of reporter proteins, with enzymatic reporters allowing for a linear detection range spanning four orders of magnitude and peptide reporters (which can be mapped to unique targets) enabling highly multiplexed visual detection. We used INSPECTR to detect a panel of five respiratory viral targets in a single reaction via a lateral-flow readout and ~4,000 copies of viral RNA via additional ambient-temperature rolling circle amplification of the expression cassette. Leveraging synthetic biology to simplify workflows for nucleic acid diagnostics may facilitate their broader applicability at the point of care.

RAD54 N-terminal domain is a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions.

In eukaryotes, RAD54 catalyzes branch migration (BM) of Holliday junctions, a basic process during DNA repair, replication, and recombination. RAD54 also stimulates RAD51 recombinase and has other activities. Here, we investigate the structural determinants for different RAD54 activities. We find that the RAD54 N-terminal domain (NTD) is responsible for initiation of BM through two coupled, but distinct steps; specific binding to Holliday junctions and RAD54 oligomerization. Furthermore, we find that the RAD54 oligomeric state can be controlled by NTD phosphorylation at S49, a CDK2 consensus site, which inhibits RAD54 oligomerization and, consequently, BM. Importantly, the effect of phosphorylation on RAD54 oligomerization is specific for BM, as it does not affect stimulation of RAD51 recombinase by RAD54. Thus, the transition of the oligomeric states provides an important control of the biological functions of RAD54 and, likely, other multifunctional proteins.

Targeting BRCA1- and BRCA2-deficient cells with RAD52 small molecule inhibitors.

RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair.