A serum-stable RNA aptamer specific for SARS-CoV-2 neutralizes viral entry.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created an urgent need for new technologies to treat COVID-19. Here we report a 2'-fluoro protected RNA aptamer that binds with high affinity to the receptor binding domain (RBD) of SARS-CoV-2 spike protein, thereby preventing its interaction with the host receptor ACE2. A trimerized version of the RNA aptamer matching the three RBDs in each spike complex enhances binding affinity down to the low picomolar range. Binding mode and specificity for the aptamer-spike interaction is supported by biolayer interferometry, single-molecule fluorescence microscopy, and flow-induced dispersion analysis in vitro. Cell culture experiments using virus-like particles and live SARS-CoV-2 show that the aptamer and, to a larger extent, the trimeric aptamer can efficiently block viral infection at low concentration. Finally, the aptamer maintains its high binding affinity to spike from other circulating SARS-CoV-2 strains, suggesting that it could find widespread use for the detection and treatment of SARS-CoV-2 and emerging variants.

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions.

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can 'slide' on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed.