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. Similar primer extension reactions performed using nucleoside triphosphate mixtures containing both C-5 modified tCTP and C-5 modified tUTP substrates enable the production of doubly modified TNA strands for a panel of 20 chemotype combinations. Kinetic measurements reveal faster on-rates (kon) and tighter binding affinity constants (Kd) for engineered versions of TNA aptamers carrying chemotypes at both pyrimidine positions as compared to their singly modified counterparts. These findings expand the chemical space of evolvable non-natural genetic polymers by offering a path for improving the quality of biologically stable TNA aptamers for future clinical applications.

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. We demonstrate the success of de novo discovery by affinity selection of threomers against the receptor binding domain of the S1 protein from SARS-CoV-2. Detailed biochemical characterization of the enriched population identified threomers with binding affinity values that are comparable to aptamers produced by conventional SELEX. This work establishes a highly parallelizable path for querying diverse chemical repertoires and may offer a viable route for accelerating the discovery of therapeutic aptamers.

Urea and Acetamide Rich Solutions Circumvent the Strand Inhibition Problem to Allow Multiple Rounds of DNA and RNA Copying.

For decades prebiotic chemists have attempted to achieve replication of RNA under prebiotic conditions with only limited success. One of the long-recognized impediments to achieving true replication of a duplex (copying of both strands) is the so-called strand inhibition problem. Specifically, while the two strands of an RNA (or DNA) duplex can be separated by heating, upon cooling the strands of a duplex will reanneal before mononucleotide or oligonucleotide substrates can bind to the individual strands. Here we demonstrate that a class of plausible prebiotic solvents, when coupled with thermal cycling and varying levels of hydration, circumvents the strand inhibition problem, and allows multiple rounds of information transfer from both strands of a duplex (replication). Replication was achieved by simultaneous ligation of oligomers that bind to their templates with the aid of the solvents. The solvents used consisted of concentrated solutions of urea and acetamide in water (UAcW), components that were likely abundant on the early Earth. The UAcW solvent system favors the annealing of shorter strands over the re-annealing of long strands, thereby circumventing strand inhibition. We observed an improvement of DNA and RNA replication yields by a factor of 100× over aqueous buffer. Information transfer in the UAcW solvent system is robust, being achieved for a range of solvent component ratios, various drying conditions, and in the absence or presence of added salts.

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. Insights into the mechanism of TNA synthesis were obtained from a high-resolution X-ray crystal structure of the postcatalytic complex bound to the primer-template duplex. A structural analysis reveals a large cavity in the enzyme active site that can accommodate the side chain of C-5-modified tUTP substrates. Our findings expand the chemical space of evolvable nucleic acid systems by providing a synthetic route to artificial genetic polymers that are uniformly modified with diversity-enhancing functional groups.

SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance.

OBJECTIVES: Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits. METHODS: We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines. RESULTS: Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 °C for 30-min and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60-180 copies/mL by absolute quantification. Furthermore, a five-sample-pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. CONCLUSION: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).

Solvent viscosity facilitates replication and ribozyme catalysis from an RNA duplex in a model prebiotic process.

The RNA World hypothesis posits that RNA was once responsible for genetic information storage and catalysis. However, a prebiotic mechanism has yet to be reported for the replication of duplex RNA that could have operated before the emergence of polymerase ribozymes. Previously, we showed that a viscous solvent enables information transfer from one strand of long RNA duplex templates, overcoming 'the strand inhibition problem'. Here, we demonstrate that the same approach allows simultaneous information transfer from both strands of long duplex templates. An additional challenge for the RNA World is that structured RNAs (like those with catalytic activity) function poorly as templates in model prebiotic RNA synthesis reactions, raising the question of how a single sequence could serve as both a catalyst and as a replication template. Here, we show that a viscous solvent also facilitates the transition of a newly synthesized hammerhead ribozyme sequence from its inactive, duplex state to its active, folded state. These results demonstrate how fluctuating environmental conditions can allow a ribozyme sequence to alternate between acting as a template for replication and functioning as a catalyst, and illustrate the potential for temporally changing environments to enable molecular processes necessary for the origin of life.