Chemical Primer Extension in Seconds

Röthlingshöfer, Manuel; Kervio, Eric; Lommel, Tanja; Plutowski, Ulrich; Hochgesand, Annette; Richert, Clemens

doi:10.1002/ange.200801260

Cited by 16 publications

(9 citation statements)

References 37 publications

(23 reference statements)

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“…In either case, the activated ribonucleotides have organic leaving groups at their 5′-phosphates, rather than the pyrophosphate leaving group of polymerase substrates. Under typical reaction conditions, elongation of strands takes hours or days, unless modified primers and/or nucleotides are used (21,22). Further, yields are often low (4,23), and the partial hydrolysis of monomers makes it difficult to copy longer sequences (24), unless primer and template are immobilized and spent monomers are removed periodically (25).…”

Section: Introductionmentioning

confidence: 99%

Enzyme-free ligation of dimers and trimers to RNA primers

Sosson

Pfeffer

Richert

2019

Nucleic Acids Research

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The template-directed formation of phosphodiester bonds between two nucleic acid components is a pivotal process in biology. To induce such a reaction in the absence of enzymes is a challenge. This challenge has been met for the extension of a primer with mononucleotides, but the ligation of short oligonucleotides (dimers or trimers) has proven difficult. Here we report a method for ligating dimers and trimers of ribonucleotides using in situ activation in aqueous buffer. All 16 different dimers and two trimers were tested. Binding studies by NMR showed low millimolar dissociation constants for complexes between representative dimers and hairpins mimicking primer–template duplexes, confirming that a weak template effect is not the cause of the poor ligating properties of these short oligomers. Rather, cyclization was found to compete with ligation, with up to 90% of dimer being converted to the cyclic form during the course of an assay. This side reaction is strongly sequence dependent and more pronounced for dimers than for trimers. Under optimized reaction conditions, high yields were observed with strongly pairing purines at the 3′-terminus. These results show that short oligomers of ribonucleotides are competent reactants in enzyme-free copying.

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Section: Introductionmentioning

confidence: 99%

Enzyme-free ligation of dimers and trimers to RNA primers

Sosson

Pfeffer

Richert

2019

Nucleic Acids Research

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“…These features support the use of templated reactions in nucleic acid detection in which product formation indicates the presence of the target structure. [2,3] Several chemical reactions have been used in a DNA-or RNA-templated fashion, including organic reactions such as ligation, [4][5][6][7][8][9][10][11] extension, [12,13] cleavage, [14][15][16][17][18] and transfer [19][20][21] reactions as well as organometal approaches. [22][23][24] The work in the field of nucleic acid diagnostics addresses two major issues, selectivity and sensitivity.…”

Section: Introductionmentioning

confidence: 99%

Nucleic Acid Templated Reactions: Consequences of Probe Reactivity and Readout Strategy for Amplified Signaling and Sequence Selectivity

Grossmann

Seitz

2009

Chemistry A European J

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DNA- and RNA-templated chemical reactions can serve as a diagnostic means for the detection of nucleic acids. Reaction schemes that allow amplified detection are of high interest for polymerase chain reaction (PCR)-free DNA and RNA diagnosis. These reactions typically draw upon the catalytic activity of the template, which is able to trigger the conversion of many signaling molecules per template molecule. However, the design of reactive probes that allow both sensitive and selective nucleic acid detection is a challenge and requires insight into three major parameters: a) reactivity of functional groups involved, b) affinity of probes for the template, and c) the readout system. In this study we used peptide nucleic acid (PNA)-based probes to investigate in detail the signaling power and the selectivity of a transfer reaction derived from a native chemical ligation. We show that subtle variations of the thioesters involved had a tremendous impact on the sensitivity and selectivity of the reaction system. The results suggest that reactions at turnover conditions require low rates of non-templated reaction pathways to provide high target selectivity and sensitivity. On the other hand, very high rates of templated reactions should be avoided to allow mismatched probe-template complexes to dissociate prior to bond formation. Furthermore, the temperature dependence of the DNA-catalyzed transfer reaction was studied and provided insight into crucial strand-exchange processes. Further improvements of selective signaling were achieved through a new readout based on pyrene-transfer reactions. This method reduces background signals and enables significant increases in the signaling rates compared with previous fluorescence-based methods.

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“…Furthermore, in our UV‐melting study of the 2′‐Te‐modified DNA duplexes, as expected, we observed T m decreases for the Te‐DNA duplexes. Since the T m decreases are dependent on the location of the Te modifications and the size of the protecting groups, Te derivatization might be a useful strategy for probing DNA and RNA polymerization and catalysis 33–35. Moreover, this Te derivatization of nucleic acids has great potential in nucleic acid X‐ray crystallography as well as in structural and functional studies of nucleic acid–protein complexes.…”

Section: Resultsmentioning

confidence: 99%

Synthesis of the First Tellurium‐Derivatized Oligonucleotides for Structural and Functional Studies

Sheng

Huang

2009

Chemistry A European J

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We report here the first synthesis of Te-nucleoside phosphoramidites and Te-modified oligonucleotides. We protected the 2'-tellurium functionality by alkylation and found that the Te functionality is compatible with solid-phase synthesis and that the Te oligonucleotides are stable during deprotection and purification. In addition, the redox properties of the Te functionalities have been explored. We found that the telluride and telluoxide DNAs are interchangeable by redox reactions. At elevated temperature, the Te-DNA can also be site-specifically fragmented oxidatively or reductively when 2'-TePh functionality is present, whereas elimination of the nucleobase is observed in the presence of 2'-TeMe. Moreover, the stability of the DNA duplexes derivatized with the Te functionalities has been investigated. Our Te derivatization of nucleic acids provides a novel approach for investigating DNA damage as well as for structure and function studies of nucleic acids and their protein complexes.

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Chemical Primer Extension in Seconds

Cited by 16 publications

References 37 publications

Enzyme-free ligation of dimers and trimers to RNA primers

Enzyme-free ligation of dimers and trimers to RNA primers

Nucleic Acid Templated Reactions: Consequences of Probe Reactivity and Readout Strategy for Amplified Signaling and Sequence Selectivity

Synthesis of the First Tellurium‐Derivatized Oligonucleotides for Structural and Functional Studies

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