Evaluating TNA stability under simulated physiological conditions

Culbertson, Michelle C.; Temburnikar, Kartik; Sau, Sujay P.; Liao, Jingwen; Bala, Saikat; Chaput, John C.

doi:10.1016/j.bmcl.2016.03.118

Cited by 75 publications

(88 citation statements)

References 22 publications

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“…This finding is consistent with previous studies demonstrating that short stretches of TNA are highly resistantt on uclease degradation. [13,30] In summary,w ed emonstrated that Bst DNA polymerase is an efficient and faithful TNA-dependent DNA polymerase with activity superior to that of SSII. We furthers howed that aT NA replication system composed of Bst and Kod-RI can be used to support the in vitro selection of TNA aptamers.…”

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confidence: 91%

“…Differences in the hydrodynamic shell between aptamer-protein complexes and free aptamer result in ar elative change of movement along the temperature gradient. [13,30] In summary,w ed emonstrated that Bst DNA polymerase is an efficient and faithful TNA-dependent DNA polymerase with activity superior to that of SSII. The strong correlation between the affinitiesm easured by dot blot and MST suggest that the K D value of aptamer T1 is~200 nm.F urthermore, aptamer T1 retained activity in the presence of RNase A, whereas bindingo fa nR NA aptamers elected by using the same strategy was lost ( Figures S7 and S8,T able S2).…”

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confidence: 96%

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Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase

Dunn

Chaput

2016

ChemBioChem

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Recent advances in polymerase engineering have enabled the replication of xenonucleic acid (XNA) polymers with backbone structures distinct from those found in nature. By introducing a selective amplification step into the replication cycle, functional XNA molecules have been isolated by in vitro selection with binding and catalytic activity. Despite these successes, coding and decoding genetic information in XNA polymers remains limited by the fidelity and catalytic efficiency of engineered XNA polymerases. In particular, the process of reverse transcribing XNA back into DNA for amplification by PCR has been problematic. Here, we show that Geobacillus stearothermophilus (Bst) DNA polymerase I functions as an efficient and faithful threose nucleic acid (TNA)-dependent DNA polymerase. Bst DNA polymerase generates ∼twofold more cDNA with threefold fewer mutations than Superscript II (SSII), which was previously the best TNA reverse transcriptase. Notably, Bst also functions under standard magnesium-dependent conditions, whereas SSII requires manganese ions to relax the enzyme's substrate specificity. We further demonstrate that Bst DNA polymerase can support the in vitro selection of TNA aptamers by evolving a TNA aptamer to human α-thrombin.

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confidence: 91%

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confidence: 96%

Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase

Dunn

Chaput

2016

ChemBioChem

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“…For instance, variants of the Thermococcus aquaticus DNAP obtained by in vitro selection can generate amplification products of up to 2 kb of fully phosphorothioate-substituted DNA (Ghadessy et al, 2004), which is resistant to exonucle-ases and can be further chemically modified with iodoacetamides. A commercial version of the archaean 9°N DNAP (Therminator, New England BioLabs) carrying a single mutation (A485L; Gardner, Joyce, & Jack, 2004) can copy DNA templates into threose nucleic acid (TNA) polymers with high fidelity (Ichida, Horhota, Zou, McLaughlin, & Szostak, 2005), generating an XNA shown to be resistant to degradation in human serum (Culbertson et al, 2016).…”

Section: Background Informationmentioning

confidence: 99%

Xenobiotic Nucleic Acid (XNA) Synthesis by Phi29 DNA Polymerase

Torres

Pinheiro

2018

CP Chemical Biology

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Phi29 DNA polymerase (DNAP) is the replicative enzyme of the Bacillus subtilis bacteriophage Phi29. Its extraordinary processivity and its ability to perform isothermal amplification of DNA are central to many molecular biology applications, including high-sensitivity detection and large-scale production of DNA. We present here Phi29 DNAP as an efficient catalyst for the production of various artificial nucleic acids (XNAs) carrying backbone modifications such as 1,5-anhydrohexitol nucleic acid (HNA), 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA), and 2'-fluoro-2'-deoxyribonucleic acid (2'-fluoro-DNA). A full protocol for the synthesis of HNA polymers by an exonuclease-deficient variant (D12A) of Phi29 DNAP plus a detailed guide for the design and test of novel XNA synthetase reactions performed by Phi29 DNAP are provided. © 2018 by John Wiley & Sons, Inc.

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“…Notably, the ability of TNAs to induce immune responses has not previously been examined. Similar to sequences composed of phosphorothioate backbones, the TNA equivalents are highly resistant to serum nucleases (13), making them an attractive tool for the future development of nucleic acid-based therapeutics. Recent advances in TNA synthesis using engineered polymerases have allowed for the selection of TNAs that target human thrombin (14) and HIV reverse transcriptase, opening the door for selections against a variety of other protein targets.…”

Section: Differential Mrna Upregulation By Low and High Doses Of Tlr9mentioning

confidence: 99%

Activation of innate immune responses by a CpG oligonucleotide sequence composed entirely of threose nucleic acid

Lange

Burke-Aguero

Chaput

2018

Preprint

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words)Recent advances in synthetic biology have led to the development of nucleic acid polymers with backbone structures distinct from those found in nature, termed xeno-nucleic acids (XNAs).Several unique properties of XNAs make them attractive as nucleic acid therapeutics, most notably their high resistance to serum nucleases and ability to form Watson-Crick base-pairing with DNA and RNA. The ability of XNAs to induce immune responses has not been investigated. Threose nucleic acid (TNA), a type of XNA, is recalcitrant to nuclease digestion and capable of undergoing Darwinian evolution to produce high affinity aptamers; thus, TNA is an attractive candidate for diverse applications, including nucleic acid therapeutics. Here, we evaluated a TNA oligonucleotide derived from a CpG oligonucleotide sequence known to activate TLR9-dependent immune signaling in B cell lines. We observed a slight induction of relevant mRNA signals, robust B cell line activation, and negligible effects on cellular proliferation.Chemical modifications have been utilized extensively to improve the binding energy, stability, and tolerability of potential therapeutics, including antisense oligonucleotides, siRNAs, and aptamers, among others. Phosphorothioate modifications in particular have been effective due to their ability to enhance cellular uptake and evade nuclease-mediated degradation in serum.More recently, advances in synthetic biology have led to the development of an expanded set of nucleic acid polymers with backbone structures distinct from those found in nature, termed xeno-nucleic acids (XNAs)(1,2). XNAs are particularly attractive in the field of nucleic acid therapeutics due to several unique properties of several XNAs, mostly notably their high resistance to serum nucleases (2) and their ability to form Watson-Crick base-pairing with DNA and RNA (1). For example, an XNA-based therapeutic for the treatment of neovascular agerelated macular degeneration, pegaptanib, was licensed by Eyetech Pharmaceuticals/Pfizer and is currently marketed under the name of Macugen (3,4). Pegaptanib is an XNA-adapted version of an RNA aptamer that binds VEGF165, the misregulation of which can cause abnormal blood vessel growth resulting in bleeding, scarring, and irreversible damage to the photoreceptors(3).2'-fluoro XNA modifications to pegaptanib were specifically included for evasion of degradation.XNA modifications are under exploration for a variety of other applications as well, including nanostructures for therapeutic delivery of payloads and diagnostics (2,4,5). Thus, XNA-based nucleic acid-based tools hold much promise in the field. However, XNAs have not been extensively evaluated for their ability to induce innate immune responses, and such evaluations will be critical for further therapeutic development of XNAs. Notably, some chemical modifications have been shown to abolish or decrease immune signaling (e.g. locked nucleic acid and 2'-Fluoro substitutions) (6), while others robustly induce immune signaling (e.g. phosphorothioate Cp...

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Evaluating TNA stability under simulated physiological conditions

Cited by 75 publications

References 22 publications

Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase

Reverse Transcription of Threose Nucleic Acid by a Naturally Occurring DNA Polymerase

Xenobiotic Nucleic Acid (XNA) Synthesis by Phi29 DNA Polymerase

Activation of innate immune responses by a CpG oligonucleotide sequence composed entirely of threose nucleic acid

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