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

Jackson, L.N.; Chim, Nicholas; Shi, Changhua; Chaput, John C.

doi:10.1093/nar/gkz513

Cited by 39 publications

(40 citation statements)

References 64 publications

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“…Non-functional sequences were removed by washing the beads with buffer and functional TNA molecules that remained bound to the ATP-coated beads were recovered by competitive elution using wash buffer that was supplemented with 5 mM ATP. The eluted TNA sequences were reverse-transcribed into cDNA using a natural DNA polymerase isolated from the bacterial species Geobacillus stearothermophilus (Bst), which is known to function with general XNA reverse transcriptase activity [ 24 , 25 ]. The reverse-transcribed DNA was then amplified by PCR to produce a population of double-stranded DNA.…”

Section: Resultsmentioning

confidence: 99%

In Vitro Selection of an ATP-Binding TNA Aptamer

Zhang

Chaput

2020

Molecules

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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.

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Section: Resultsmentioning

confidence: 99%

In Vitro Selection of an ATP-Binding TNA Aptamer

Zhang

Chaput

2020

Molecules

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“…We obtained the first structural insights into an enzyme with XNA reverse transcriptase activity by solving crystal structures of Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of FANA and TNA (Fig. 14) (Jackson et al ., 2019). Comparison of these structures with Bst DNA polymerase bound to the natural DNA primer–template duplex (Chim et al ., 2018) reveals differences, particularly at the enzyme active site as well as in protein interactions with the duplexes (Jackson et al ., 2019).…”

Section: Promiscuous Activities Of Natural Polymerasesmentioning

confidence: 99%

Engineering polymerases for applications in synthetic biology

Nikoomanzar

Chim

Yik

et al. 2020

Quart. Rev. Biophys.

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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.

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“…Identification of enzymes capable of reverse transcribing XNAs demonstrates the potential of these divergent chemistries as genetic materials and is crucial for in vitro selections [ 156 ]. Some XNAs can be reverse transcribed by natural polymerases (e.g., TNA and FANA by Bst polymerase [ 157 – 159 ]). Alternately, engineering approaches must be taken [ 79 , 88 ].…”

Section: Templated Synthesis Of Non-natural Nucleic Acidsmentioning

confidence: 99%

Modified nucleic acids: replication, evolution, and next-generation therapeutics

2020

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Modified nucleic acids, also called xeno nucleic acids (XNAs), offer a variety of advantages for biotechnological applications and address some of the limitations of first-generation nucleic acid therapeutics. Indeed, several therapeutics based on modified nucleic acids have recently been approved and many more are under clinical evaluation. XNAs can provide increased biostability and furthermore are now increasingly amenable to in vitro evolution, accelerating lead discovery. Here, we review the most recent discoveries in this dynamic field with a focus on progress in the enzymatic replication and functional exploration of XNAs.

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

Cited by 39 publications

References 64 publications

In Vitro Selection of an ATP-Binding TNA Aptamer

In Vitro Selection of an ATP-Binding TNA Aptamer

Engineering polymerases for applications in synthetic biology

Modified nucleic acids: replication, evolution, and next-generation therapeutics

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