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.

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.

A Tool for the Import of Natural and Unnatural Nucleoside Triphosphates into Bacteria.

Nucleoside triphosphates play a central role in biology, but efforts to study these roles have proven difficult because the levels of triphosphates are tightly regulated in a cell and because individual triphosphates can be difficult to label or modify. In addition, many synthetic biology efforts are focused on the development of unnatural nucleoside triphosphates that perform specific functions in the cellular environment. In general, both of these efforts would be facilitated by a general means to directly introduce desired triphosphates into cells. Previously, we demonstrated that recombinant expression of a nucleoside triphosphate transporter from Phaeodactylum tricornutum (PtNTT2) in Escherichia coli functions to import triphosphates that are added to the media. Here, to explore the generality and utility of this approach, we report a structure-activity relationship study of PtNTT2. Using a conventional competitive uptake inhibition assay, we characterize the effects of nucleobase, sugar, and triphosphate modification, and then develop an LC-MS/MS assay to directly measure the effects of the modifications on import. Lastly, we use the transporter to import radiolabeled or 2'-fluoro-modified triphosphates and quantify their incorporation into DNA and RNA. The results demonstrate the general utility of the PtNTT2-mediated import of natural or modified nucleoside triphosphates for different molecular or synthetic biology applications.

Synthesis and Evolution of a Threose Nucleic Acid Aptamer Bearing 7-Deaza-7-Substituted Guanosine Residues.

In vitro selection experiments carried out on artificial genetic polymers require robust and faithful methods for copying genetic information back and forth between DNA and xeno-nucleic acids (XNA). Previously, we have shown that Kod-RI, an engineered polymerase developed to transcribe DNA templates into threose nucleic acid (TNA), can function with high fidelity in the absence of manganese ions. However, the transcriptional efficiency of this enzyme diminishes greatly when individual templates are replaced with libraries of DNA sequences, indicating that manganese ions are still required for in vitro selection. Unfortunately, the presence of manganese ions in the transcription mixture leads to the misincorporation of tGTP nucleotides opposite dG residues in the templating strand, which are detected as G-to-C transversions when the TNA is reverse transcribed back into DNA. Here we report the synthesis and fidelity of TNA replication using 7-deaza-7-modified guanosine base analogues in the DNA template and incoming TNA nucleoside triphosphate. Our findings reveal that tGTP misincorporation occurs via a Hoogsteen base pair in which the incoming tGTP residue adopts a syn conformation with respect to the sugar. Substitution of tGTP for 7-deaza-7-phenyl tGTP enabled the synthesis of TNA polymers with >99% overall fidelity. A TNA library containing the 7-deaza-7-phenyl guanine analogue was used to evolve a biologically stable TNA aptamer that binds to HIV reverse transcriptase with low nanomolar affinity.

Synthesis of 2'-Deoxy-α-l-threofuranosyl Nucleoside Triphosphates.

α-l-Threofuranosyl nucleic acid (TNA) is an artificial genetic polymer in which the natural five-carbon ribose sugar found in RNA has been replaced with an unnatural four-carbon threose sugar. Despite a different sugar-phosphate backbone, TNA is capable of forming stable, antiparallel Watson-Crick duplex structures with itself and with complementary strands of DNA and RNA. This property of intersystem base pairing, coupled with the chemical simplicity of threose relative to ribose, provides support for TNA as a candidate RNA progenitor in the evolution of life. In an effort to evaluate the functional properties of TNA by in vitro evolution, engineered polymerases have been developed that are capable of copying information back and forth between DNA and TNA. However, the current generation of TNA polymerases function with reduced activity relative to their natural counterparts, which limits the evaluation of TNA as a primordial genetic material. Here, we describe the chemical synthesis and polymerase recognition of 2'-deoxy-α-l-threofuranosyl nucleoside 3'-triphosphates (dtNTPs) as chain-terminating reagents in a polymerase-mediated TNA synthesis reaction. The synthesis of dtNTPs should make it possible to investigate the mechanism of TNA synthesis by X-ray crystallography by trapping the polymerase in the catalytically active conformation.

A parallel stranded G-quadruplex composed of threose nucleic acid (TNA).

G-rich sequences can adopt four-stranded helical structures, called G-quadruplexes, that self-assemble around monovalent cations like sodium (Na+ ) and potassium (K+ ). Whether similar structures can be formed from xeno-nucleic acid (XNA) polymers with a shorter backbone repeat unit is an unanswered question with significant implications on the fold space of functional XNA polymers. Here, we examine the potential for TNA (α-l-threofuranosyl nucleic acid) to adopt a four-stranded helical structure based on a planar G-quartet motif. Using native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) and solution-state nuclear magnetic resonance (NMR) spectroscopy, we show that despite a backbone repeat unit that is one atom shorter than the backbone repeat unit found in DNA and RNA, TNA can self-assemble into stable G-quadruplex structures that are similar in thermal stability to equivalent DNA structures. However, unlike DNA, TNA does not appear to discriminate between Na+ and K+ ions, as G-quadruplex structures form equally well in the presence of either ion. Together, these findings demonstrate that despite a shorter backbone repeat unit, TNA is capable of self-assembling into stable G-quadruplex structures.

Synthesis of α-l-Threofuranosyl Nucleoside 3'-Monophosphates, 3'-Phosphoro(2-Methyl)imidazolides, and 3'-Triphosphates.

α-l-Threofuranosyl nucleic acid (TNA) is an artificial genetic polymer composed of vicinal 2',3'-phosphodiester bonds linking adjacent threofuranosyl nucleosides. TNA is one of a small number of genetic polymers that are both highly resistant to nuclease digestion and capable of cross-pairing with DNA and RNA. Although an efficient method for synthesizing TNA nucleosides has been reported, very few advances have been made in the synthesis of phosphorylated TNA compounds. Here, we describe a highly efficient method for synthesizing α-l-threofuranosyl nucleoside 3'-monophosphates (tNMPs), 3'-phosphoro(2-methyl)imidazolides (2-MeImptNs), and 3'-triphosphates (tNTPs) bearing the four genetic bases of adenine (A), cytosine (C), thymine (T), and guanine (G). We suggest that this strategy, which provides access to grams of tNMPs, hundreds of milligrams of 2-MeImptNs, and tens of milligrams of tNTPs, will help advance the use of TNA monomers in exobiology and biotechnology applications.

Structural Insights into Conformation Differences between DNA/TNA and RNA/TNA Chimeric Duplexes.

Threose nucleic acid (TNA) is an artificial genetic polymer capable of heredity and evolution, and is studied in the context of RNA chemical etiology. It has a four-carbon threose backbone in place of the five-carbon ribose of natural nucleic acids, yet forms stable antiparallel complementary Watson-Crick homoduplexes and heteroduplexes with DNA and RNA. TNA base-pairs more favorably with RNA than with DNA but the reason is unknown. Here, we employed NMR, ITC, UV, and CD to probe the structural and dynamic properties of heteroduplexes of RNA/TNA and DNA/TNA. The results indicate that TNA templates the structure of heteroduplexes, thereby forcing an A-like helical geometry. NMR measurement of kinetic and thermodynamic parameters for individual base pair opening events reveal unexpected asymmetric "breathing" fluctuations of the DNA/TNA helix. The results suggest that DNA is unable to fully adapt to the conformational constraints of the rigid TNA backbone and that nucleic acid breathing dynamics are determined from both backbone and base contributions.

A Scalable Synthesis of α-L-Threose Nucleic Acid Monomers.

Recent advances in polymerase engineering have made it possible to copy information back and forth between DNA and artificial genetic polymers composed of TNA (α-L-threofuranosyl-(3',2') nucleic acid). This property, coupled with enhanced nuclease stability relative to natural DNA and RNA, warrants further investigation into the structural and functional properties of TNA as an artificial genetic polymer for synthetic biology. Here, we report a highly optimized chemical synthesis protocol for constructing multigram quantities of TNA nucleosides that can be readily converted to nucleoside 2'-phosphoramidites or 3'-triphosphates for solid-phase and polymerase-mediated synthesis, respectively. The synthetic protocol involves 10 chemical transformations with three crystallization steps and a single chromatographic purification, which results in an overall yield of 16-23% depending on the identity of the nucleoside (A, C, G, T).

Evaluating TNA stability under simulated physiological conditions.

Chemically modified oligonucleotides are routinely used as diagnostic and therapeutic agents due to their enhanced biological stability relative to natural DNA and RNA. Here, we examine the biological stability of α-l-threofuranosyl nucleic acid (TNA), an artificial genetic polymer composed of repeating units of α-l-threofuranosyl sugars linked by 2',3'-phosphodiester bonds. We show that TNA remains undigested after 7days of incubation in the presence of either 50% human serum or human liver microsomes and is stable against snake venom phosphordiesterase (a highly active 3' exonuclease). We further show that TNA will protect internal DNA residues from nuclease digestion and shield complementary RNA strands from RNA degrading enzymes. Together, these results demonstrate that TNA is an RNA analogue with high biological stability.

Multistep divergent synthesis of benzimidazole linked benzoxazole/benzothiazole via copper catalyzed domino annulation.

An efficient, facile synthesis of structurally diverse benzimidazole integrated benzoxazole and benzothiazoles has been developed. In a multi-step synthetic sequence, 4-fluoro-3-nitrobenzoic acid was converted into benzimidazole bis-heterocycles, via the intermediacy of benzimidazole linked ortho-chloro amines. The amphiphilic reactivity of this intermediate was designed to achieve the title compounds by the reaction of various acid chlorides and isothiocyanates in a single step through the in situ formation of ortho-chloro anilides and thioureas under microwave irradiation. A versatile one pot domino annulation reaction was developed to involve the reaction of benzimidazole linked ortho-chloro amines with acid chlorides and isothiocyanates. The initial acylation and urea formation followed by copper catalyzed intramolecular C-O and C-S cross coupling reactions furnished the angularly oriented bis-heterocycles which bear a close resemblance to the streptomyces antibiotic UK-1.

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.