Chemical Primer Extension: Efficiently Determining Single Nucleotides in DNA

Hagenbuch, Patrizia; Kervio, Eric; Hochgesand, Annette; Plutowski, Ulrich; Richert, Clemens

doi:10.1002/ange.200501794

Cited by 43 publications

(69 citation statements)

References 27 publications

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“…The 3′-amino terminus of the primer gives increased nucleophilicity toward oxyazabenzotriazolides (OAt) (26) as monomers (Fig. 1, 3 and 4).…”

Section: Resultsmentioning

confidence: 99%

“…For the kinetic studies, only the monomer complementary to the template base was used to keep the system as simple as possible. During the first phase of our study, we employed a downstream-binding oligonucleotide that provides additional stacking surface to the monomer (Scheme 1A) (26).…”

Section: Resultsmentioning

confidence: 99%

“…* The rates of the fastest reaction (TCTas core template) and the slowest reaction (CAG as core template) differed by a factor of 81, i.e., by less than two orders of magnitude. The hydrolysis of the most slowly reacting monomer (dTMP-OAt) has a half-life time of 16.2 h in assay buffer (26). It is thus more than one order of magnitude slower than even the slowest of the primer extensions (template CAG, t 1∕2 ¼ 80 min).…”

Section: Resultsmentioning

confidence: 99%

“…We measured rates of incorporation and fidelity on DNA templates possessing any of the 64 possible combinations of templating base and neighboring residues. Our study was made feasible by the availability of chemically activated monomers (26), amino primers (27) (Fig. 1, 3), and mass spectrometrically monitored assays for quantitative monitoring (28), unbiased by the effect of labels (29).…”

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confidence: 99%

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Templating efficiency of naked DNA

Kervio

Hochgesand

Steiner

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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111

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Template-directed synthesis of complementary strands is pivotal for life. Nature employs polymerases for this reaction, leaving the ability of DNA itself to direct the incorporation of individual nucleotides at the end of a growing primer difficult to assess. Using 64 sequences, we now find that any of the four nucleobases, in combination with any neighboring residue, support enzyme-free primer extension when primer and mononucleotide are sufficiently reactive, with ≥93% primer extension for all sequences. Between the 64 possible base triplets, the rate of extension for the poorest template, CAG, with A as templating base, and the most efficient template, TCT, with C as templating base, differs by less than two orders of magnitude. Further, primer extension with a balanced mixture of monomers shows ≥72% of the correct extension product in all cases, and ≥90% incorporation of the correct base for 46 out of 64 triplets in the presence of a downstream-binding strand. A mechanism is proposed with a binding equilibrium for the monomer, deprotonation of the primer, and two chemical steps, the first of which is most strongly modulated by the sequence. Overall, rates show a surprisingly smooth reactivity landscape, with similar incorporation on strongly and weakly templating sequences. These results help to clarify the substrate contribution to copying, as found in polymerase-catalyzed replication, and show an important feature of DNA as genetic material.

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“…The 3′-amino terminus of the primer gives increased nucleophilicity toward oxyazabenzotriazolides (OAt) (26) as monomers (Fig. 1, 3 and 4).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Templating efficiency of naked DNA

Kervio

Hochgesand

Steiner

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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111

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show abstract

“…The potentially prebiotic synthesis of imidazoles under primitive Earth conditions has been investigated (7). On the other hand, Richert and coworkers reported the use of benzotriazole-activated monomers to improve the rate of primer extension (8). In an alternative approach, the in situ activation of monoribonucleotides, and subsequent template-guided polymerization, has been achieved by Richert and coworkers (9), using a carbodiimide reagent together with N-alkyl-imidazole catalysts.…”

mentioning

confidence: 99%

Insight into the mechanism of nonenzymatic RNA primer extension from the structure of an RNA-GpppG complex

Zhang

Tam

Walton

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The nonenzymatic copying of RNA templates with imidazoleactivated nucleotides is a well-studied model for the emergence of RNA self-replication during the origin of life. We have recently discovered that this reaction can proceed through the formation of an imidazolium-bridged dinucleotide intermediate that reacts rapidly with the primer. To gain insight into the relationship between the structure of this intermediate and its reactivity, we cocrystallized an RNA primer-template complex with a close analog of the intermediate, the triphosphate-bridged guanosine dinucleotide GpppG, and solved a high-resolution X-ray structure of the complex. The structure shows that GpppG binds the RNA template through two Watson-Crick base pairs, with the primer 3ʹ-hydroxyl oriented to attack the 5ʹ-phosphate of the adjacent G residue. Thus, the GpppG structure suggests that the bound imidazoliumbridged dinucleotide intermediate would be preorganized to react with the primer by in-line S N 2 substitution. The structures of bound GppG and GppppG suggest that the length and flexibility of the 5ʹ-5ʹ linkage are important for optimal preorganization of the complex, whereas the position of the 5ʹ-phosphate of bound pGpG explains the slow rate of oligonucleotide ligation reactions. Our studies provide a structural interpretation for the observed reactivity of the imidazolium-bridged dinucleotide intermediate in nonenzymatic RNA primer extension.RNA self-replication | diguanosine dinucleotide | crystal structure | origin of life I n the RNA world hypothesis, the emergence of RNA-catalyzed RNA replication is thought to have been preceded by a stage in which RNA replication was driven purely through chemical processes (1, 2). The nonenzymatic RNA-templated polymerization of activated nucleotides or oligonucleotides has been extensively studied, with the intent of optimizing the rate, extent, and fidelity of nonenzymatic RNA/DNA polymerization (3, 4). Numerous phosphate-activating groups have been studied in the context of nonenzymatic RNA replication. For example, imidazoles such as 2-methylimidazole (5) and, more recently, 2-aminoimidazole (6), have been found to be useful phosphate activators. The potentially prebiotic synthesis of imidazoles under primitive Earth conditions has been investigated (7). On the other hand, Richert and coworkers reported the use of benzotriazole-activated monomers to improve the rate of primer extension (8). In an alternative approach, the in situ activation of monoribonucleotides, and subsequent template-guided polymerization, has been achieved by Richert and coworkers (9), using a carbodiimide reagent together with N-alkyl-imidazole catalysts.Many of the thermodynamic and kinetic parameters associated with nonenzymatic RNA replication have been quantitatively determined (10-14). Until recently, the general assumption has been that nonenzymatic primer extension with activated mononucleotides involves classical S N 2 nucleophilic substitution, in which the nucleophilic 3ʹ-hydroxyl group of the primer attack...

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Nonenzymatic RNA replication in a mixture of ‘spent’ nucleotides

Patki,

Rajamani

2023

FEBS Letters

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Nonenzymatic template‐directed replication would have been affected by co‐solutes in a heterogeneous prebiotic soup due to lack of enzymatic machinery. Unlike in contemporary biology, these reactions use chemically activated nucleotides, which undergo rapid hydrolysis forming nucleoside monophosphates (‘spent’ monomers). These co‐solutes cannot extend the primer but continue to base pair with the template, thereby interfering with replication. We, therefore, aimed to understand how a mixture of ‘spent’ ribonucleotides would affect nonenzymatic replication. We observed the inhibition of replication in the mixture, wherein the predominant contribution came from the cognate Watson–Crick monomer, showing potential sequence dependence. Our study highlights how nonenzymatic RNA replication would have been directly affected by co‐solutes, with ramifications for the emergence of functional polymers in an RNA World.

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Chemical Primer Extension: Efficiently Determining Single Nucleotides in DNA

Cited by 43 publications

References 27 publications

Templating efficiency of naked DNA

Templating efficiency of naked DNA

Insight into the mechanism of nonenzymatic RNA primer extension from the structure of an RNA-GpppG complex

Nonenzymatic RNA replication in a mixture of ‘spent’ nucleotides

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