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. Here, we design REVEALR into a novel genotyping assay that detects single-base mismatches corresponding to each of the major SARS-CoV-2 strains found in the United States. Of 34 sequence-verified patient samples collected in early, mid, and late 2021 at the UCI Medical Center in Orange, California, REVEALR identified the correct variant [Wuhan-Hu-1, alpha (B.1.1.7), gamma (P.1), epsilon (B.1.427/9), delta (B.1.617.2), and omicron (B.1.1.529)] with 100% accuracy. The assay, which is programmable and amenable to multiplexing, offers an important new approach to personalized diagnostics.

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 in vitro 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. Kinetic measurements reveal that the side chain modifications are critical for generating threomers with slow off-rate binding kinetics. These findings expand the chemical space of evolvable non-natural genetic systems to include functional groups that enhance protein target binding by mimicking the structural properties of traditional antibodies.

P(V) Reagents for the Scalable Synthesis of Natural and Modified Nucleoside Triphosphates.

Natural and modified nucleoside triphosphates impact nearly every major aspect of healthcare research from DNA sequencing to drug discovery. However, a scalable synthetic route to these molecules has long been hindered by the need for purification by high performance liquid chromatography (HPLC). Here, we describe a fundamentally different approach that uses a novel P(V) pyrene pyrophosphate reagent to generate derivatives that are purified by silica gel chromatography and converted to the desired compounds on scales vastly exceeding those achievable by HPLC. The power of this approach is demonstrated through the synthesis of a broad range of natural and unnatural nucleoside triphosphates (dNTPs and xNTPs) using protocols that are efficient, inexpensive, and operationally straightforward.

Evolution of a General RNA-Cleaving FANA Enzyme.

The isolation of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that catalysis is not limited to natural biopolymers, but it remains unknown whether such systems can achieve robust catalysis with Michaelis-Menten kinetics. Here, we describe an efficient RNA-cleaving 2'-fluoroarabino nucleic acid enzyme (FANAzyme) that functions with a rate enhancement of >106-fold over the uncatalyzed reaction and exhibits substrate saturation kinetics typical of most natural enzymes. The FANAzyme was generated by in vitro evolution using natural polymerases that were found to recognize FANA substrates with high fidelity. The enzyme comprises a small 25 nucleotide catalytic domain flanked by substrate-binding arms that can be engineered to recognize diverse RNA targets. Substrate cleavage occurs at a specific phosphodiester bond located between an unpaired guanine and a paired uracil in the substrate recognition arm. Our results expand the chemical space of nucleic acid enzymes to include nuclease-resistant scaffolds with strong catalytic activity.