Revealr-Based Diagnostic Panel for Rapid Detection of Acute Respiratory Infections.

RNA-encoded viral nucleic acid analyte reporter (REVEALR) is a rapid and highly sensitive point-of-care diagnostic developed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, genotyping, and quantification. Here, we extend the breadth of this nucleic acid technology to include a viral respiratory panel that can detect low attomolar levels of influenza A (IAV), influenza B (IBV), SARS-CoV-2 (CoV2), and the respiratory syncytial virus (RSV). Of 39 clinical samples collected at the UCI Medical Center in Orange, California, the extended REVEALR panel showed a positive predictive agreement and negative predictive agreement of 100% for IAV, CoV2, and RSV in sequence-verified clinical samples, with 0 false positive results.

DNA Replication across α-l-(3'-2')-Threofuranosyl Nucleotides Mediated by Human DNA Polymerase η.

α-l-(3'-2')-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with -alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product.

Structure and Stability of Ago2 MID-Nucleotide Complexes: All-in-One (Drop) His-SUMO Tag Removal, Nucleotide Binding, and Crystal Growth.

The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5'-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa.

An improved synthesis of guanosine TNA phosphoramidite for oligonucleotide synthesis.

The chemical synthesis of guanosine nucleosides generates both the and regioisomers, which require careful separation to obtain the desired isomer. To preferentially obtain the isomer, a bulky diphenylcarbamoyl (DPC) group can be installed at the position of guanine. However, installation of the DPC group presents a challenging task due to low solubility of the -acetyl protected guanine.

Improved synthesis and polymerase recognition of 7-deaza-7-modified α-l-threofuranosyl guanosine analogs.

Threofuranosyl nucleic acid (TNA), an artificial genetic polymer known for its nuclease resistance and acid stability, has grown in popularity as a genetically-encoded material for applications in synthetic biology and biomedicine. TNA oligonucleotide synthesis requires enzymatic or solid phase synthesis pathways that rely on monomer building blocks that are not commercially available and can only be obtained by chemical synthesis. Here we present a synthetic route to 7-deaza-7-modified tGTP and phosphoramidite analogs that is operationally simpler than our previously described strategy.

Increasing the functional density of threose nucleic acid.

Chemical strategies that augment genetic polymers with amino acid residues that are overrepresented on the paratope surface of an antibody offer a promising route for enhancing the binding properties of nucleic acid aptamers. Here, we describe the chemical synthesis of α-l-threofuranosyl cytidine nucleoside triphosphate (tCTP) carrying either a benzyl or phenylpropyl side chain at the pyrimidine C-5 position. Polymerase recognition studies indicate that both substrates are readily incorporated into a full-length α-l-threofuranosyl nucleic acid (TNA) product by extension of a DNA primer-template duplex with an engineered TNA polymerase.

Functionally Enhanced XNA Aptamers Discovered by Parallelized Library Screening.

In vitro evolution strategies have been used for >30 years to generate nucleic acid aptamers against therapeutic targets of interest, including disease-associated proteins. However, this process requires many iterative cycles of selection and amplification, which severely restricts the number of target and library design combinations that can be explored in parallel. Here, we describe a single-round screening approach to aptamer discovery that relies on function-enhancing chemotypes to increase the distribution of high-affinity sequences in a random-sequence library.

Parameterizing the Binding Properties of XNA Aptamers Isolated from a Low Stringency Selection.

Machine learning offers a guided approach to aptamer discovery, but more information is needed to develop algorithms that can intelligently identify high-performing aptamers to a broad array of targets. Critical to this effort is the need to experimentally parameterize the difference between low and high affinity binders to a given target. Although classical selection experiments help define the upper limit by converging on a small number of tight binding sequences, very little is known about the lower limit of binding that defines the boundary between binders and nonbinders.

Engineering TNA polymerases through iterative cycles of directed evolution.

DNA polymerases are important tools for biotechnology, synthetic biology, and chemical biology as they are routinely used to amplify and edit genetic information. However, natural polymerases do not recognize artificial genetic polymers (also known as xeno-nucleic acids or XNAs) with unique sugar-phosphate backbone structures. Directed evolution offers a possible solution to this problem by facilitating the discovery of engineered versions of natural polymerases that can copy genetic information back and forth between DNA and XNA.

Stability and mechanism of threose nucleic acid toward acid-mediated degradation.

Xeno-nucleic acids (XNAs) have gained significant interest as synthetic genetic polymers for practical applications in biomedicine, but very little is known about their biophysical properties. Here, we compare the stability and mechanism of acid-mediated degradation of α-l-threose nucleic acid (TNA) to that of natural DNA and RNA. Under acidic conditions and elevated temperature (pH 3.

Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs.

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'--methyl or 2'-fluoro ribose modifications.

Highly Parallelized Screening of Functionally Enhanced XNA Aptamers in Uniform Hydrogel Particles.

Xeno-nucleic acid (XNA) aptamers based on evolvable non-natural genetic polymers hold enormous potential as future diagnostic and therapeutic agents. However, time-consuming and costly procedures requiring the purification of individual XNA sequences produced by large-scale polymerase-mediated primer extension reactions pose a major bottleneck to the discovery of highly active XNA motifs for biomedical applications. Here, we describe a straightforward approach for rapidly surveying the binding properties of XNA aptamers identified by in vitro selection.

Chemical evolution of an autonomous DNAzyme with allele-specific gene silencing activity.

Low activity has been the primary obstacle impeding the use of DNA enzymes (DNAzymes) as gene silencing agents in clinical applications. Here we describe the chemical evolution of a DNAzyme with strong catalytic activity under near physiological conditions. The enzyme achieves ~65 turnovers in 30 minutes, a feat only previously witnessed by the unmodified parent sequence under forcing conditions of elevated Mg and pH.

Metabolic adjustments in response to ATP spilling by the small DX protein in a strain.

ATP wasting is recognized as an efficient strategy to enhance metabolic activity and productivity of specific metabolites in several microorganisms However, such strategy has been rarely implemented in species whereas antibiotic production by members of this genus is known to be triggered in condition of phosphate limitation that is correlated with a low ATP content. In consequence, to assess the effects of ATP spilling on the primary and specialized metabolisms of , the gene encoding the small synthetic protein DX, that has high affinity for ATP and dephosphorylates ATP into ADP, was cloned in the integrative vector pOSV10 under the control of the strong E promoter. This construct and the empty vector were introduced into the species yielding A37 and A36, respectively.

Amplification-Free COVID-19 Detection by Digital Droplet REVEALR.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, exposed a pressing need for new public health tools for pathogen detection, disease diagnosis, and viral genotyping. REVEALR (RNA-encoded viral nucleic acid analyte reporter) is an isothermal DNAzyme-based point-of-care diagnostic that functions with a detection limit of ∼10 copies/μL when coupled with a preamplification step and can be utilized for viral genotyping of SARS-CoV-2 variants of concern through base pair mismatch recognition in a competitive binding format. Here, we describe an advanced REVEALR platform, termed digital droplet REVEALR (ddREVEALR), that can achieve direct viral detection and absolute sample quantitation utilizing a signal amplification strategy that relies on chemical modifications, DNAzyme multiplexing, and volume compression.

Crystallographic analysis of engineered polymerases synthesizing phosphonomethylthreosyl nucleic acid.

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with backbone structures composed of non-ribose or non-deoxyribose sugars. Phosphonomethylthreosyl nucleic acid (pTNA), a type of XNA that does not base pair with DNA or RNA, has been suggested as a possible genetic material for storing synthetic biology information in cells. A critical step in this process is the synthesis of XNA episomes using laboratory-evolved polymerases to copy DNA information into XNA.

REVEALR-Based Genotyping of SARS-CoV-2 Variants of Concern in Clinical Samples.

The SARS-CoV-2 virus has evolved into new strains that increase viral transmissibility and reduce vaccine protection. The rapid circulation of these more harmful strains across the globe has created a pressing need for alternative public health screening tools. REVEALR (RNA-encoded viral nucleic acid analytic reporter), a rapid and highly sensitive DNAzyme-based detection system, functions with perfect accuracy against patient-derived clinical samples.

Evolution of Functionally Enhanced α-l-Threofuranosyl Nucleic Acid Aptamers.

Synthetic genetic polymers (xeno-nucleic acids, XNAs) have the potential to transition aptamers from laboratory tools to therapeutic agents, but additional functionality is needed to compete with antibodies. Here, we describe the evolution of a biologically stable artificial genetic system composed of α-l-threofuranosyl nucleic acid (TNA) that facilitates the production of backbone- and base-modified aptamers termed "threomers" that function as high quality protein capture reagents. Threomers were discovered against two prototypical protein targets implicated in human diseases through a combination of selection and next-generation sequencing using uracil nucleotides that are uniformly equipped with aromatic side chains commonly found in the paratope of antibody-antigen crystal structures.

Synthesis and Polymerase Recognition of Threose Nucleic Acid Triphosphates Equipped with Diverse Chemical Functionalities.

Expanding the chemical space of evolvable non-natural genetic polymers (XNAs) to include functional groups that enhance protein target binding affinity offers a promising route to therapeutic aptamers with high biological stability. Here we describe the chemical synthesis and polymerase recognition of 10 chemically diverse functional groups introduced at the C-5 position of α-l-threofuranosyl uridine nucleoside triphosphate (tUTP). We show that the set of tUTP substrates is universally recognized by the laboratory-evolved polymerase Kod-RSGA.

Transliteration of synthetic genetic enzymes.

Functional nucleic acids lose activity when their sequence is prepared in the backbone architecture of a different genetic polymer. The only known exception to this rule is a subset of aptamers whose binding mechanism involves G-quadruplex formation. We refer to such examples as transliteration-a synthetic biology concept describing cases in which the phenotype of a nucleic acid molecule is retained when the genotype is written in a different genetic language.

REVEALR: A Multicomponent XNAzyme-Based Nucleic Acid Detection System for SARS-CoV-2.

Isothermal amplification strategies capable of rapid, inexpensive, and accurate nucleic acid detection provide new options for large-scale pathogen detection, disease diagnosis, and genotyping. Here we report a highly sensitive multicomponent XNA-based nucleic acid detection platform that combines analyte preamplification with X10-23-mediated catalysis to detect the viral pathogen responsible for COVID-19. The platform, termed RNA-Encoded Viral Nucleic Acid Analyte Reporter (REVEALR), functions with a detection limit of ≤20 aM (∼10 copies/μL) using conventional fluorescence and paper-based lateral flow readout modalities.

Functional Comparison of Laboratory-Evolved XNA Polymerases for Synthetic Biology.

Artificial genetic polymers (XNAs) have enormous potential as new materials for synthetic biology, biotechnology, and molecular medicine; yet, very little is known about the biochemical properties of XNA polymerases that have been developed to synthesize and reverse-transcribe XNA polymers. Here, we compare the substrate specificity, thermal stability, reverse transcriptase activity, and fidelity of laboratory-evolved polymerases that were established to synthesize RNA, 2'-fluoroarabino nucleic acid (FANA), arabino nucleic acid (ANA), hexitol nucleic acid (HNA), threose nucleic acid (TNA), and phosphonomethylthreosyl nucleic acid (PMT). We find that the mutations acquired to facilitate XNA synthesis increase the tolerance of the enzymes for sugar-modified substrates with some sacrifice to protein-folding stability.

Following replicative DNA synthesis by time-resolved X-ray crystallography.

The mechanism of DNA synthesis has been inferred from static structures, but the absence of temporal information raises longstanding questions about the order of events in one of life's most central processes. Here we follow the reaction pathway of a replicative DNA polymerase using time-resolved X-ray crystallography to elucidate the order and transition between intermediates. In contrast to the canonical model, the structural changes observed in the time-lapsed images reveal a catalytic cycle in which translocation precedes catalysis.

A biologically stable DNAzyme that efficiently silences gene expression in cells.

Efforts to use RNA-cleaving DNA enzymes (DNAzymes) as gene-silencing agents in therapeutic applications have stalled due to their low efficacy in clinical trials. Here we report a xeno-nucleic-acid-modified version of the classic DNAzyme 10-23 that achieves multiple-turnover activity under cellular conditions and resists nuclease digestion. The new reagent, X10-23, overcomes the problem of product inhibition, which limited previous 10-23 designs, using molecular chemotypes with DNA, 2'-fluoroarabino nucleic acid and α-L-threofuranosyl nucleic acid backbone architectures that balance the effects of enhanced biological stability with RNA hybridization and divalent metal ion coordination.