Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′‐5′ Linkages

Cozens, Christopher; Mutschler, Hannes; Nelson, Geoffrey M.; Houlihan, Gillian; Taylor, Alexander I.; Holliger, Philipp

doi:10.1002/anie.201508678

Cited by 24 publications

(18 citation statements)

References 38 publications

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“…Recently, Holliger and co-workers (Cozens et al, 2015 ) engineered the polymerase TGLLK (TgoT: Y409G, I521L, F545L, E664K), which proved to be effective at fully replacing purine dNTPs and NTPs with their respective 3′-deoxy or 3′- O -methyl analogs with defined 2′-5′ linkages allowing template-directed synthesis and reverse transcription of oligonucleotides with mixed 2′-5′/3′-5′ backbone linkages, expanding the repertoire for future in vitro evolution experiments.…”

Section: Polymerases For Nucleotides Bearing Sugar Modificationsmentioning

confidence: 99%

Modified Nucleoside Triphosphates for In-vitro Selection Techniques

2016

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The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed.

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Section: Polymerases For Nucleotides Bearing Sugar Modificationsmentioning

confidence: 99%

Modified Nucleoside Triphosphates for In-vitro Selection Techniques

2016

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“…Although there were slight differences in efficiency, generally SFM4‐3, SFM4‐6, and SFM4‐9 were more active (Figure S4). This is the first study to evaluate Taq mutants for this activity, and it showed that they can synthesize a 2′ linkage; previously only one Tgo mutant had been shown to synthesize 2′ linkages . These experiments open the door to future engineering of Taq mutants for the synthesis of M‐DNA with 2′ linkages.…”

Section: Resultsmentioning

confidence: 79%

“…Interestingly, one of the modified nucleotides that was recognized with higher efficiency had a 3′ modification (3′=OMe); 3′‐modified nucleotides with reversible protecting groups have applications in high‐throughput sequencing and in synthesizing RNA with 2′ linkage . There is only one report of a Taq mutant that recognizes a 3′‐protected nucleotide; no Taq mutant recognizes 3′‐modified nucleotides with 2′OH modifications.…”

Section: Resultsmentioning

confidence: 99%

Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates

et al. 2017

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Chemical modifications can enhance the properties of DNA by imparting nuclease resistance and generating more-diverse physical structures. However, native DNA polymerases generally cannot synthesize significant lengths of DNA with modified nucleotide triphosphates. Previous efforts have identified a mutant of DNA polymerase I from Thermus aquaticus DNA (SFM19) as capable of synthesizing a range of short, 2'-modified DNAs; however, it is limited in the length of the products it can synthesize. Here, we rationally designed and characterized ten mutants of SFM19. From this, we identified enzymes with substantially improved activity for the synthesis of 2'F-, 2'OH-, 2'OMe-, and 3'OMe-modified DNA as well as for reverse transcription of 2'OMe DNA. We also evaluated mutant DNA polymerases previously only tested for synthesis for 2'OMe DNA and showed that they are capable of an expanded range of modified DNA synthesis. This work significantly expands the known combinations of modified DNA and Taq DNA polymerase mutants.

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“…Primer-dependent RNA synthesis has a number of applications, as it obviates the need to initiate transcription with a 5 ′ -pppG and allows a wide range of chemical groups to be added during solid-phase synthesis of primers (e.g., divergent 5 ′ -cap chemistries and cap-proximal epigenetic modification such as N 6 ,2 ′ -O-dimethyladenosine [m 6 A m ] and 7-methylguanosine [m 7 G] [reviewed in Meyer and Jaffrey 2017]). Further engineering enabled incorporation of regioisomeric 2 ′ -5 ′ linkages into both DNA and RNA by replacement of purine dNTPs with 3 ′ -deoxy or 3 ′ -O-methyl (3 ′ OMe) analogs (Cozens et al 2015).…”

Section: Engineering Primer-dependent Rna Polymerasesmentioning

confidence: 99%

Beyond DNA and RNA: The Expanding Toolbox of Synthetic Genetics

Taylor

Houlihan

Holliger

2019

Cold Spring Harb Perspect Biol

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The remarkable physicochemical properties of the natural nucleic acids, DNA and RNA, define modern biology at the molecular level and are widely believed to have been central to life's origins. However, their ability to form repositories of information as well as functional structures such as ligands (aptamers) and catalysts (ribozymes/DNAzymes) is not unique. A range of nonnatural alternatives, collectively termed xeno nucleic acids (XNAs), are also capable of supporting genetic information storage and propagation as well as evolution. This gives rise to a new field of "synthetic genetics," which seeks to expand the nucleic acid chemical toolbox for applications in both biotechnology and molecular medicine. In this review, we outline XNA polymerase and reverse transcriptase engineering as a key enabling technology and summarize the application of "synthetic genetics" to the development of aptamers, enzymes, and nanostructures.

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Enzymatic Synthesis of Nucleic Acids with Defined Regioisomeric 2′‐5′ Linkages

Cited by 24 publications

References 38 publications

Modified Nucleoside Triphosphates for In-vitro Selection Techniques

Modified Nucleoside Triphosphates for In-vitro Selection Techniques

Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates

Beyond DNA and RNA: The Expanding Toolbox of Synthetic Genetics

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