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 3.3 at 90°C), TNA was found to be significantly more resistant to acid-mediated degradation than DNA and RNA. Mechanistic insights gained by reverse-phase HPLC and mass spectrometry indicate that the resilience of TNA toward low pH environments is due to a slower rate of depurination caused by induction of the 2'-phosphodiester linkage. Similar results observed for 2',5'-linked DNA and 2'-O-methoxy-RNA implicate the position of the phosphodiester group as a key factor in destabilizing the formation of the oxocarbenium intermediate responsible for depurination and strand cleavage of TNA. Biochemical analysis indicates that strand cleavage occurs by ß-elimination of the 2'-phosphodiester linkage to produce an upstream cleavage product with a 2'-threose sugar and a downstream cleavage product with a 3' terminal phosphate. This work highlights the unique physicochemical properties available to evolvable non-natural genetic polymers currently in development for biomedical applications.

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. Here, we investigate the polymerase recognition of pTNA nucleotides using X-ray crystallography to capture the post-catalytic complex of engineered polymerases following the sequential addition of two pTNA nucleotides onto the 3'-end of a DNA primer. High-resolution crystal structures reveal that the polymerase mediates Watson-Crick base pairing between the extended pTNA adducts and the DNA template. Comparative analysis studies demonstrate that the sugar conformation and backbone position of pTNA are structurally more similar to threose nucleic acid than DNA even though pTNA and DNA share the same six-atom backbone repeat length. Collectively, these findings provide new insight into the structural determinants that guide the enzymatic synthesis of an orthogonal genetic polymer, and may lead to the discovery of new variants that function with enhanced activity.

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. Here, we apply a quantitative approach to explore the diversity of aptamers isolated from two identical in vitro selections performed under low stringency conditions. Starting from a library of 1 trillion unique threose nucleic acid (TNA) sequences, 7 rounds of selection were performed to enrich binders to a known aptagenic target. High density sequencing of each round of selection followed by a detailed kinetic analysis of 136 TNA aptamers yielded a narrow range of equilibrium dissociation constants (KD = ∼ 1-15 nM) that were consistent between two experimental replicates. These findings offer insights into the lower limit of binding that may be expected for aptamers generated against aptagenic targets and could provide useful constraints for evaluating the results of experimental and computational approaches.

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.

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'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.

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. Here, we extend the concept of transliteration to include nucleic acid enzymes (XNAzymes) that mediate site-specific cleavage of an RNA substrate. We show that an in vitro selected 2'-fluoroarabino nucleic acid (FANA) enzyme retains catalytic activity when its sequence is prepared as α-l-threofuranosyl nucleic acid (TNA), and vice versa, a TNA enzyme that remains functional when its sequence is prepared as FANA. Structure probing with DMS supports the hypothesis that FANA and TNA enzymes having the same primary sequence can adopt similarly folded tertiary structures. These findings provide new insight into the sequence-structure-function paradigm governing biopolymer folding.

Structural interpretation of the effects of threo-nucleotides on nonenzymatic template-directed polymerization.

The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here, we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3'-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer 10-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands.

Engineering polymerases for applications in synthetic biology.

DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.

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.

Redesigning the Genetic Polymers of Life.

Genomes can be viewed as constantly updated memory systems where information propagated in cells is refined over time by natural selection. This process, commonly known as heredity and evolution, has been the sole domain of DNA since the origin of prokaryotes. Now, some 3.5 billion years later, the pendulum of discovery has swung in a new direction, with carefully trained practitioners enabling the replication and evolution of "xeno-nucleic acids" or "XNAs"-synthetic genetic polymers in which the natural sugar found in DNA and RNA has been replaced with a different type of sugar moiety. XNAs have attracted significant attention as new polymers for synthetic biology, biotechnology, and medicine because of their unique physicochemical properties that may include increased biological stability, enhanced chemical stability, altered helical geometry, or even elevated thermodynamics of Watson-Crick base pairing.This Account describes our contribution to the field of synthetic biology, where chemical synthesis and polymerase engineering have allowed my lab and others to extend the concepts of heredity and evolution to synthetic genetic polymers with backbone structures that are distinct from those found in nature. I will begin with a discussion of α-l-threofuranosyl nucleic acid (TNA), a specific type of XNA that was chosen as a model system to represent any XNA system. I will then proceed to discuss advances in organic chemistry that were made to enable the synthesis of gram quantities of TNA phosphoramidites and nucleoside triphosphates, the monomers used for solid-phase and polymerase-mediated TNA synthesis, respectively. Next, I will recount our development of droplet-based optical sorting (DrOPS), a single-cell microfluidic technique that was established to evolve XNA polymerases in the laboratory. This section will conclude with structural insights that have been gained by solving X-ray crystal structures of a laboratory-evolved TNA polymerase and a natural DNA polymerase that functions with general reverse transcriptase activity on XNA templates.The final passage of this Account will examine the role that XNAs have played in synthetic biology by highlighting examples in which engineered polymerases have enabled the evolution of biologically stable affinity reagents (aptamers) and catalysts (XNAzymes) as well as the storage and retrieval of binary information encoded in electronic word and picture file formats. Because these examples provide only a glimpse of what the future may have in store for XNA, I will conclude the Account with my thoughts on how synthetic genetic polymers could help drive new innovations in synthetic biology and molecular medicine.

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. With a total assay time of 1 h, REVEALR provides a convenient nucleic acid alternative to equivalent CRISPR-based approaches, which have become popular methods for SARS-CoV-2 detection. The assay shows no cross-reactivity for other in vitro transcribed respiratory viral RNAs and functions with perfect accuracy against COVID-19 patient-derived clinical samples.

Allele-Specific RNA Knockdown with a Biologically Stable and Catalytically Efficient XNAzyme.

Therapeutic targeting of allele-specific single nucleotide mutations in RNA is a major challenge in biology and medicine. Herein, we describe the utility of the XNAzyme X10-23 to knock down allele-specific mRNA sequences in cells. We demonstrate the value of this approach by targeting the "undruggable" mutation G12V in oncogenic KRAS. Our results demonstrate how catalytic XNAs could be employed to suppress the expression of mRNAs carrying disease-causing mutations that are difficult to target at the protein level with small molecule therapeutics.

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.

Synthesis and polymerase recognition of a pyrrolocytidine TNA triphosphate.

Synthetic genetics is an area of synthetic biology that aims to extend the properties of heredity and evolution to artificial genetic polymers, commonly known as xeno-nucleic acids or XNAs. In addition to establishing polymerases that are able to convert genetic information back and forth between DNA and XNA, efforts are underway to construct XNAs with expanded chemical functionality. α-L-Threose nucleic acid (TNA), a type of XNA that is recalcitrant to nuclease digestion and amenable to Darwinian evolution, provides a model system for developing XNAs with functional groups that are not present in natural DNA and RNA. Here, we describe the synthesis and polymerase activity of a cytidine TNA triphosphate analog (6-phenyl-pyrrolocytosine, tCp TP) that maintains Watson-Crick base pairing with guanine. Polymerase-mediated primer extension assays show that tCp TP is an efficient substrate for Kod-RI, a DNA-dependent TNA polymerase developed to explore the functional properties of TNA by in vitro selection. Fidelity studies reveal that a cycle of TNA synthesis and reverse transcription occurs with 99.9% overall fidelity when tCp TP and 7-deaza-tGTP are present as TNA substrates. This result expands the toolkit of TNA building blocks available for in vitro selection.

Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase.

Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2'-deoxy-2'-fluoro-ß-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.

Generating Biologically Stable TNA Aptamers that Function with High Affinity and Thermal Stability.

Aptamers are often prone to nuclease digestion, which limits their utility in many biomedical applications. Here we describe a xeno-nucleic acid system based on α-l-threofuranosyl nucleic acid (TNA) that is completely refractory to nuclease digestion. The use of an engineered TNA polymerase permitted the isolation of functional TNA aptamers that bind to HIV reverse transcriptase (HIV RT) with KD's of ∼0.4-4.0 nM. The aptamers were identified using a display strategy that provides a powerful genotype-phenotype linkage. The TNA aptamers remain active in the presence of nuclease and exhibit markedly higher thermal stability than monoclonal antibodies. The combined properties of biological stability, high binding affinity, and thermal stability make TNA aptamers a powerful system for the development of diagnostic and therapeutic agents.

Orthogonal Genetic Systems.

Xenobiology is an emerging area of synthetic biology that aims to safeguard genetically engineered cells by storing synthetic biology information in xeno-nucleic acid polymers (XNAs). Critical to the success of this effort is the need to establish cellular systems that can maintain an XNA chromosome in actively dividing cells. This viewpoint discusses the structural parameters of the nucleic acid backbone that should be considered when designing an orthogonal genetic system that can replicate without interference from the endogenous genome. In addition to practical value, these studies have the potential to provide new fundamental insight into the structure and function properties of unnatural nucleic acid polymers.

Evaluating the Catalytic Potential of a General RNA-Cleaving FANA Enzyme.

The discovery of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that natural genetic polymers are not unique in their ability to function as enzymes. However, all known examples of in vitro selected XNA enzymes function with lower activity than their natural counterparts, suggesting that XNAs might be limited in their ability to fold into structures with high catalytic activity. To explore this problem, we evaluated the catalytic potential of FANAzyme 12-7, an RNA-cleaving catalyst composed entirely of 2'-fluoroarabino nucleic acid (FANA) that was evolved to cleave RNA at a specific phosphodiester bond located between an unpaired guanine and a paired uracil in the substrate recognition arm. Here, we show that this activity extends to chimeric DNA substrates that contain a central riboguanosine (riboG) residue at the cleavage site. Surprisingly, FANAzyme 12-7 rivals known DNAzymes that were previously evolved to cleave chimeric DNA substrates under physiological conditions. These data provide convincing evidence that FANAzyme 12-7 maintains the catalytic potential of equivalent DNAzymes, which has important implications for the evolution of XNA catalysts and their contributions to future applications in synthetic biology.

In Vitro Selection of an ATP-Binding TNA Aptamer.

Recent advances in polymerase engineering have made it possible to isolate aptamers from libraries of synthetic genetic polymers (XNAs) with backbone structures that are distinct from those found in nature. However, nearly all of the XNA aptamers produced thus far have been generated against protein targets, raising significant questions about the ability of XNA aptamers to recognize small molecule targets. Here, we report the evolution of an ATP-binding aptamer composed entirely of α-L-threose nucleic acid (TNA). A chemically synthesized version of the best aptamer sequence shows high affinity to ATP and strong specificity against other naturally occurring ribonucleotide triphosphates. Unlike its DNA and RNA counterparts that are susceptible to nuclease digestion, the ATP-binding TNA aptamer exhibits high biological stability against hydrolytic enzymes that rapidly degrade DNA and RNA. Based on these findings, we suggest that TNA aptamers could find widespread use as molecular recognition elements in diagnostic and therapeutic applications that require high biological stability.

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.