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Brakmann, Susanne; Grzeszik, Sandra

doi:10.1002/1439-7633(20010302)2:3<153::aid-cbic153>3.3.co;2-2

Cited by 3 publications

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“…[36][37][38][39] The generation of functionalized nucleic acids for in vitro selection involves the enzymatic polymerization of synthetically functionalized nucleoside triphosphates (dXTPs in which X is any given nucleobase). [40] An abiding interest in this approach is highlighted in numerous studies in which synthetic nucleotides have been successfully incorporated for the discovery of highly functionalized nucleic acids, [41][42][43][44][45] aptamers, [46][47][48][49][50][51] RNA-based enzymes [52,53] and DNAzymes. [54][55][56][57] Our initial attempt at enhancing the chemical repertoire of DNAzymes resulted in the DNAzyme Dz9 25 -11, which required both the imidazole and amine-bearing modified nucleotides 1 and 2 (Scheme 1) for activity.…”

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confidence: 99%

A DNAzyme with Three Protein‐Like Functional Groups: Enhancing Catalytic Efficiency of M²⁺‐Independent RNA Cleavage

et al. 2009

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[a] Catalytically efficient, sequence-specific RNA cleavage holds great therapeutic value for selective gene inactivation against viral infection and cancer. Towards that end, SELEX and related combinatorial methods of in vitro selection [1][2][3] have been used to discover RNA-cleaving DNAzymes that have received considerable attention. [4][5][6][7][8] In addition to therapeutic use in catalysing sequence-specific destruction of mRNA for targeted gene deactivation, applications of catalysis to sensing have also been suggested. [4,[9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] Like ribozymes, DNAzymes present a limited chemical repertoire compared to proteins. [27,28] [33] These findings highlight an inextricable link between high concentrations of Mg 2 + and catalytic efficiency. [34,35] By the same token, one must hypothesise that the scarcity of intracellular Mg 2 + limits the efficacy of DNAzymes in vivo.This catalytic shortcoming might be circumvented by selecting for DNAzymes with synthetically appended chemical functionalities that enhance the chemical repertoire of nucleic acid enzymes, which is otherwise impaired by a low physiological concentration of M 2 + . [36][37][38][39] The generation of functionalized nucleic acids for in vitro selection involves the enzymatic polymerization of synthetically functionalized nucleoside triphosphates (dXTPs in which X is any given nucleobase).[40] An abiding interest in this approach is highlighted in numerous studies in which synthetic nucleotides have been successfully incorporated for the discovery of highly functionalized nucleic acids, [41][42][43][44][45] aptamers, [46][47][48][49][50][51] RNA-based enzymes [52,53] and DNAzymes. [54][55][56][57] Our initial attempt at enhancing the chemical repertoire of DNAzymes resulted in the DNAzyme Dz9 25 -11, which required both the imidazole and amine-bearing modified nucleotides 1 and 2 (Scheme 1) for activity.[28] Nevertheless, catalytic properties remained modest: whereas a reasonably fast rate of selfcleavage was observed (k obs~0 .3 min À1 ), the optimum temperature for self cleavage remained depressed at 13 8C; further, although the cis-cleaving DNAzyme was engineered to cleave at a single ribonucleoside linkage with both multiple turnover and high sequence specificity, [58,59] both optimal temperature (25 8C) and k cat (0.03 min À1) also remained rather low. Notably, attempts to reselect 2nd-generation catalysts from either 20 or 40 degenerate positions failed to provide any improvement and instead Dz9 25 -11 and closely related sequence variants embedded within the N40 region were selected.[60] This "null result" led to an important conclusion: modified dNTPs 1 and 2 found in Dz9 25 -11 failed to provide more efficient catalysts, even when selections were attempted from longer libraries composed of 40 nucleotides (N40). Consequently, we hypothesised that further increasing chemical diversity by introducing a third modified nucleotide bearing a cationic guanidinium ion would increase...

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confidence: 99%

A DNAzyme with Three Protein‐Like Functional Groups: Enhancing Catalytic Efficiency of M²⁺‐Independent RNA Cleavage

et al. 2009

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“…The DNA produced was analyzed by qPCR with Q5 DNA polymerase or by PCR and PAGE, which revealed efficient product formation, but not in control reactions lacking SFM4-3 or Superscript III (Figure S2). Sequencing of the amplified DNA (see Methods, Figure S2) revealed that the major mutations in both cases were A to G transitions (Table S3) and a mutation frequency for the combined transcription−reverse transcription-PCR of 2 × 10 −3 for RNA (which is only ∼30-fold reduced relative to T7 RNAPmediated transcription 13 ) and 1.2 × 10 −2 for 2′-F-C,U-RNA. This suggests that the fidelity of SFM4-3 is likely sufficient for many practical applications.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

Chen

Romesberg

2017

J. Am. Chem. Soc.

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There is increasing demand for RNA and modified RNA oligonucleotides, but in contrast to DNA oligonucleotides, they are typically prohibitively expensive to chemically synthesize, and unlike longer RNAs, they are only inefficiently produced by in vitro transcription, especially when modified. To address these challenges, we previously reported the evolution of a thermostable DNA polymerase, SFM4-3, that more efficiently accepts substrates with 2'-substituents. We now show that SFM4-3 efficiently transcribes RNA or 2'-F-modified RNA and that it also efficiently PCR amplifies oligonucleotides of mixed RNA and DNA composition. In addition, with thermocycling and the use of a novel DNA template, we demonstrate a polymerase chain transcription (PCT) reaction that results in the exponential production of orders of magnitude more RNA or modified RNA than is available by conventional transcription. PCT is more efficient and general than conventional transcription and can produce large amounts of any RNA or modified RNA oligonucleotide at a fraction of the cost of chemical synthesis.

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“…For example, bulkier substituents might not be able to accommodate to the proximity of the enzyme active site. However, a recent report demonstrated incorporation of 58 tetramethylrhodamine‐labeled dUTP‐residues in a template‐directed polymerization assay catalyzed by an exonuclease‐deficient Klenow fragment 61,62…”

Section: Single Molecule Sequencingmentioning

confidence: 99%

Functional tuning of nucleic acids by chemical modifications: Tailored oligonucleotides as drugs, devices, and diagnostics

Verma

Jäger

Thum

et al. 2003

The Chemical Record

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Chemical modifications of nucleic acids present vast opportunities for extending the functions and properties of these biomolecules. In general, efforts invested in this direction pertain to the introduction of reactive functional groups for further derivatizations of oligonucleotides with numerous reporter groups and for equipping nucleic acids with catalytic chemical moieties. This review deals with representative chemical modifications in the nucleobases, sugars, and the phosphate ester backbone and their application from novel catalytic RNA selection to nucleic acid-based biosensors.

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Cited by 3 publications

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A DNAzyme with Three Protein‐Like Functional Groups: Enhancing Catalytic Efficiency of M²⁺‐Independent RNA Cleavage

A DNAzyme with Three Protein‐Like Functional Groups: Enhancing Catalytic Efficiency of M²⁺‐Independent RNA Cleavage

Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

Functional tuning of nucleic acids by chemical modifications: Tailored oligonucleotides as drugs, devices, and diagnostics

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Cited by 3 publications

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A DNAzyme with Three Protein‐Like Functional Groups: Enhancing Catalytic Efficiency of M2+‐Independent RNA Cleavage

A DNAzyme with Three Protein‐Like Functional Groups: Enhancing Catalytic Efficiency of M2+‐Independent RNA Cleavage

Polymerase Chain Transcription: Exponential Synthesis of RNA and Modified RNA

Functional tuning of nucleic acids by chemical modifications: Tailored oligonucleotides as drugs, devices, and diagnostics

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A DNAzyme with Three Protein‐Like Functional Groups: Enhancing Catalytic Efficiency of M²⁺‐Independent RNA Cleavage

A DNAzyme with Three Protein‐Like Functional Groups: Enhancing Catalytic Efficiency of M²⁺‐Independent RNA Cleavage