Pentaphosphorylation via the Anhydride of Dihydrogen Pentametaphosphate: Access to Nucleoside Hexa- and Heptaphosphates and Study of Their Interaction with Ribonuclease A.

Pentametaphosphate is the little studied cyclic pentamer of the metaphosphate ion, [PO3]5 5-. We show that the doubly protonated form of this pentamer can be selectively dehydrated to provide the anhydride [P5O14]3- (1). This trianion is the well-defined condensed phosphate component of a novel reagent for attachment of a pentaphosphate chain to biomolecules all in one go. Here, we demonstrate by extending adenosine monophosphate (AMP) and uridine monophosphate (UMP) to their corresponding nucleoside hexaphosphates, while adenosine diphosphate (ADP) and uridine diphosphate (UDP) are phosphate chain-extended to the corresponding nucleoside heptaphosphates. Such constructs are of interest for their potential biological function with respect to RNA-processing enzymes. Thus, we go on to investigate in detail the interaction of the polyanionic constructs with ribonuclease A, a model protein containing a polycationic active site and for which X-ray crystal structures are relatively straightforward to obtain. This work presents a combined experimental and quantum chemical approach to understanding the interactions of RNase A with the new nucleoside hexa- and heptaphosphate constructs.

Sensitive detection of SARS-CoV-2 main protease 3CLpro with an engineered ribonuclease zymogen.

Alongside vaccines and antiviral therapeutics, diagnostic tools are a crucial aid in combating the COVID-19 pandemic caused by the etiological agent SARS-CoV-2. All common assays for infection rely on the detection of viral sub-components, including structural proteins of the virion or fragments of the viral genome. Selective pressure imposed by human intervention of COVID-19 can, however, induce viral mutations that decrease the sensitivity of diagnostic assays based on biomolecular structure, leading to an increase in false-negative results. In comparison, mutations are unlikely to alter the function of viral proteins, and viral machinery is under less selective pressure from vaccines and therapeutics. Accordingly, diagnostic assays that rely on biomolecular function can be more robust than ones that rely on biopolymer structure. Toward this end, we used a split intein to create a circular ribonuclease zymogen that is activated by the SARS-CoV-2 main protease, 3CLpro . Zymogen activation by 3CLpro leads to a >300-fold increase in ribonucleolytic activity, which can be detected with a highly sensitive fluorogenic substrate. This coupled assay can detect low nanomolar concentrations of 3CLpro within a timeframe comparable to that of common antigen-detection protocols. More generally, the concept of detecting a protease by activating a ribonuclease could be the basis of diagnostic tools for other indications.

Bayesian Inference Elucidates the Catalytic Competency of the SARS-CoV-2 Main Protease 3CLpro.

The main protease of SARS-CoV-2, 3CLpro, is a dimeric enzyme that is indispensable to viral replication and presents an attractive opportunity for therapeutic intervention. Previous reports regarding the key properties of 3CLpro and its highly similar SARS-CoV homologue conflict dramatically. Values of the dimeric Kd and enzymic kcat/KM differ by 106- and 103-fold, respectively. Establishing a confident benchmark of the intrinsic capabilities of this enzyme is essential for combating the current pandemic as well as potential future outbreaks. Here, we use enzymatic methods to characterize the dimerization and catalytic efficiency of the authentic protease from SARS-CoV-2. Specifically, we use the rigor of Bayesian inference in a Markov Chain Monte Carlo analysis of progress curves to circumvent the limitations of traditional Michaelis-Menten initial rate analysis. We report that SARS-CoV-2 3CLpro forms a dimer at pH 7.5 that has Kd = 16 ± 4 nM and is capable of catalysis with kcat = 9.9 ± 1.5 s-1, KM = 0.23 ± 0.01 mM, and kcat/KM = (4.3 ± 0.7) × 104 M-1 s-1. We also find that enzymatic activity decreases substantially in solutions of high ionic strength, largely as a consequence of impaired dimerization. We conclude that 3CLpro is a more capable catalyst than appreciated previously, which has important implications for the design of antiviral therapeutic agents that target 3CLpro.

Semisynthesis of Human Ribonuclease-S.

Since its conception, the ribonuclease S complex (RNase S) has led to historic discoveries in protein chemistry, enzymology, and related fields. Derived by the proteolytic cleavage of a single peptide bond in bovine pancreatic ribonuclease (RNase A), RNase S serves as a convenient and reliable model system for incorporating unlimited functionality into an enzyme. Applications of the RNase S system in biomedicine and biotechnology have, however, been hindered by two shortcomings: (1) the bovine-derived enzyme could elicit an immune response in humans, and (2) the complex is susceptible to dissociation. Here, we have addressed both limitations in the first semisynthesis of an RNase S conjugate derived from human pancreatic ribonuclease and stabilized by a covalent interfragment cross-link. We anticipate that this strategy will enable unprecedented applications of the "RNase-S" system.