Development and implementation of a simple and rapid extraction-free saliva SARS-CoV-2 RT-LAMP workflow for workplace surveillance.

Effective management of the COVID-19 pandemic requires widespread and frequent testing of the population for SARS-CoV-2 infection. Saliva has emerged as an attractive alternative to nasopharyngeal samples for surveillance testing as it does not require specialized personnel or materials for its collection and can be easily provided by the patient. We have developed a simple, fast, and sensitive saliva-based testing workflow that requires minimal sample treatment and equipment. After sample inactivation, RNA is quickly released and stabilized in an optimized buffer, followed by reverse transcription loop-mediated isothermal amplification (RT-LAMP) and detection of positive samples using a colorimetric and/or fluorescent readout. The workflow was optimized using 1,670 negative samples collected from 172 different individuals over the course of 6 months. Each sample was spiked with 50 copies/µL of inactivated SARS-CoV-2 virus to monitor the efficiency of viral detection. Using pre-defined clinical samples, the test was determined to be 100% specific and 97% sensitive, with a limit of detection of 39 copies/mL. The method was successfully implemented in a CLIA laboratory setting for workplace surveillance and reporting. From April 2021-February 2022, more than 30,000 self-collected samples from 755 individuals were tested and 85 employees tested positive mainly during December and January, consistent with high infection rates in Massachusetts and nationwide.

A catalogue of biochemically diverse CRISPR-Cas9 orthologs.

Bacterial Cas9 nucleases from type II CRISPR-Cas antiviral defence systems have been repurposed as genome editing tools. Although these proteins are found in many microbes, only a handful of variants are used for these applications. Here, we use bioinformatic and biochemical analyses to explore this largely uncharacterized diversity. We apply cell-free biochemical screens to assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of 79 Cas9 proteins, thus identifying at least 7 distinct gRNA classes and 50 different PAM sequence requirements. PAM recognition spans the entire spectrum of T-, A-, C-, and G-rich nucleotides, from single nucleotide recognition to sequence strings longer than 4 nucleotides. Characterization of a subset of Cas9 orthologs using purified components reveals additional biochemical diversity, including both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for long stretches of homology between gRNA and DNA target. Our results expand the available toolset of RNA-programmable CRISPR-associated nucleases.

Therminator DNA Polymerase: Modified Nucleotides and Unnatural Substrates.

A variant of 9°N DNA polymerase [Genbank ID (AAA88769.1)] with three mutations (D141A, E143A, A485L) and commercialized under the name "Therminator DNA polymerase" has the ability to incorporate a variety of modified nucleotide classes. This Review focuses on how Therminator DNA Polymerase has enabled new technologies in synthetic biology and DNA sequencing. In addition, we discuss mechanisms for increased modified nucleotide incorporation.

Rapid incorporation kinetics and improved fidelity of a novel class of 3'-OH unblocked reversible terminators.

Recent developments of unique nucleotide probes have expanded our understanding of DNA polymerase function, providing many benefits to techniques involving next-generation sequencing (NGS) technologies. The cyclic reversible termination (CRT) method depends on efficient base-selective incorporation of reversible terminators by DNA polymerases. Most terminators are designed with 3'-O-blocking groups but are incorporated with low efficiency and fidelity. We have developed a novel class of 3'-OH unblocked nucleotides, called Lightning Terminators™, which have a terminating 2-nitrobenzyl moiety attached to hydroxymethylated nucleobases. A key structural feature of this photocleavable group displays a 'molecular tuning' effect with respect to single-base termination and improved nucleotide fidelity. Using Therminator DNA polymerase, we demonstrate that these 3'-OH unblocked terminators exhibit superior enzymatic performance compared to two other reversible terminators, 3'-O-amino-TTP and 3'-O-azidomethyl-TTP. Lightning Terminators show maximum incorporation rates (k(pol)) that range from 35 to 45 nt/s, comparable to the fastest NGS chemistries, yet with catalytic efficiencies (k(pol)/K(D)) comparable to natural nucleotides. Pre-steady-state kinetic studies of thymidine analogs revealed that the major determinant for improved nucleotide selectivity is a significant reduction in k(pol) by >1000-fold over TTP misincorporation. These studies highlight the importance of structure-function relationships of modified nucleotides in dictating polymerase performance.

Polbase: a repository of biochemical, genetic and structural information about DNA polymerases.

Polbase (http://polbase.neb.com) is a freely accessible database of DNA polymerases and related references. It has been developed in a collaborative model with experts whose contributions reflect their varied backgrounds in genetics, structural biology and biochemistry. Polbase is designed to compile detailed results of polymerase experimentation, presenting them in a dynamic view to inform further research. After validation, results from references are displayed in context with relevant experimental details and are always traceable to their source publication. Polbase is connected to other resources, including PubMed, UniProt and the RCSB Protein Data Bank, to provide multi-faceted views of polymerase knowledge. In addition to a simple web interface, Polbase data is exposed for custom analysis by external software. With the contributions of many polymerase investigators, Polbase has become a powerful research tool covering most important aspects of polymerases, from sequence and structure to biochemistry.

Biochemical characterization of a structure-specific resolving enzyme from Sulfolobus islandicus rod-shaped virus 2.

Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C-80°C) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal requirements for four-way junction cleavage, and substrate specificity were determined. SIRV2 Hjr cleaves four-way DNA junctions with a preference for cleavage of exchange strand pairs, in contrast to host-derived resolving enzymes, suggesting fundamental differences in substrate recognition and cleavage among closely related Sulfolobus resolving enzymes. Unlike other viral resolving enzymes, such as T4 endonuclease VII or T7 endonuclease I, that cleave branched DNA replication intermediates, SIRV2 Hjr cleavage is specific to four-way DNA junctions and inactive on other branched DNA molecules. In addition, a specific interaction was detected between SIRV2 Hjr and the SIRV2 virion body coat protein (SIRV2gp26). Based on this observation, a model is proposed linking SIRV2 Hjr genome resolution to viral particle assembly.

Characterization of Sulfolobus islandicus rod-shaped virus 2 gp19, a single-strand specific endonuclease.

The hyperthermophilic Sulfolobus islandicus rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein (SIRV2gp19) annotated as a hypothetical protein with sequence homology to the RecB nuclease superfamily. Even though SIRV2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, SIRV2gp19 has not been functionally characterized. To define the minimal requirements for activity, SIRV2gp19 was purified and tested under varying conditions. SIRV2gp19 is a single-strand specific endonuclease that requires Mg(2+) for activity and is inactive on double-stranded DNA. A conserved aspartic acid in RecB nuclease superfamily Motif II (D89) is also essential for SIRV2gp19 activity and mutation to alanine (D89A) abolishes activity. Therefore, the SIRV2gp19 cleavage mechanism is similar to previously described RecB nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease activity could play a role in host chromosome degradation during SIRV2 lytic infection.

Comparative kinetics of nucleotide analog incorporation by vent DNA polymerase.

Comparative kinetic and structural analyses of a variety of polymerases have revealed both common and divergent elements of nucleotide discrimination. Although the parameters for dNTP incorporation by the hyperthermophilic archaeal Family B Vent DNA polymerase are similar to those previously derived for Family A and B DNA polymerases, parameters for analog incorporation reveal alternative strategies for discrimination by this enzyme. Discrimination against ribonucleotides was characterized by a decrease in the affinity of NTP binding and a lower rate of phosphoryl transfer, whereas discrimination against ddNTPs was almost exclusively due to a slower rate of phosphodiester bond formation. Unlike Family A DNA polymerases, incorporation of 9-[(2-hydroxyethoxy)methyl]X triphosphates (where X is adenine, cytosine, guanine, or thymine; acyNTPs) by Vent DNA polymerase was enhanced over ddNTPs via a 50-fold increase in phosphoryl transfer rate. Furthermore, a mutant with increased propensity for nucleotide analog incorporation (Vent(A488L) DNA polymerase) had unaltered dNTP incorporation while displaying enhanced nucleotide analog binding affinity and rates of phosphoryl transfer. Based on kinetic data and available structural information from other DNA polymerases, we propose active site models for dNTP, ddNTP, and acyNTP selection by hyperthermophilic archaeal DNA polymerases to rationalize structural and functional differences between polymerases.

Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases.

The nucleotide-binding site in a variety of DNA polymerases was probed by analyzing incorporation of dideoxy and acyclic chain terminators. Family B archaeon DNA polymerases Vent, Deep Vent, 9 degrees N and Pfu incorporated acyclic in preference to dideoxy terminators, while the Family A DNA polymerases Taq and Klenow preferred dideoxy terminators. These divergent biases suggest that significant differences exist in sugar recognition in these two classes of polymerases. Mutants of Vent (A488L) and Taq (F667Y) that increase incorporation of dideoxy terminators maintained the acyclic/dideoxy bias of the parent enzyme, while more efficiently incorporating both dideoxy and acyclic terminators. The preference of archaeon DNA polymerases for acyclic analogs was exploited in chain terminator DNA sequence and genotype analysis. This technology was additionally aided by identification of specific dye-modified bases that improve terminator incorporation over that of the unmodified terminator. These three enhancing effects, (i) acyclic analogs, (ii) archaeon variants and (iii) specific dyes, appear to act additively and independently to increase terminator incorporation efficiency, collectively enhancing incorporation approximately 8000-fold over the wild-type incorporation of dideoxynucleotides. Fluorescent chain terminator DNA sequence traces demonstrate the applicability of these advances in improving terminator incorporation, as required in DNA sequence and genotype determinations.