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.

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.

Reading and Writing Digital Information in TNA.

DNA has become a popular soft material for low energy, high-density information storage, but it is susceptible to damage through oxidation, pH, temperature, and nucleases in the environment. Here, we describe a new molecular chemotype for data archiving based on the unnatural genetic framework of α-l-threofuranosyl nucleic acid (TNA). Using a simple genetic coding strategy, 23 kilobytes of digital information were stored in DNA-primed TNA oligonucleotides and recovered with perfect accuracy after exposure to biological nucleases that destroyed equivalent DNA messages. We suggest that these results extend the capacity for nucleic acids to function as a soft material for low energy, high-density information storage by providing a safeguard against information loss caused by nuclease digestion.

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.

Ligase-Mediated Threose Nucleic Acid Synthesis on DNA Templates.

Ligases are a class of enzymes that catalyze the formation of phosphodiester bonds between an oligonucleotide donor with a 5' terminal phosphate and an oligonucleotide acceptor with a 3' terminal hydroxyl group. Here, we wished to explore the substrate specificity of naturally occurring DNA and RNA ligases to determine whether the molecular recognition of these enzymes is sufficiently general to synthesize alternative genetic polymers with backbone structures that are distinct from those found in nature. We chose threose nucleic acid (TNA) as a model system, as TNA is known to be biologically stable and capable of undergoing Darwinian evolution. Enzyme screening and reaction optimization identified several ligases that can recognize TNA as either the donor or acceptor strand with DNA. Less discrimination occurs on the acceptor strand indicating that the determinants of substrate specificity depend primarily on the composition of the donor strand. Remarkably, T3 and T7 ligases were able to join TNA homopolymers together, which is surprising given that the TNA backbone is one atom shorter than that of DNA. In this reaction, the base composition of the ligation junction strongly favors the formation of A-T and A-G linkages. We suggest that these results will enable the assembly of TNA oligonucleotides of lengths beyond what is currently possible by solid-phase synthesis and provide a starting point for further optimization by directed evolution.