Chemical Primer Extension: Efficiently Determining Single Nucleotides in DNA

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

doi:10.1002/anie.200501794

Cited by 67 publications

(76 citation statements)

References 28 publications

(22 reference statements)

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“…On the DNA template, the identity of the 5′ activated leaving group did not greatly affect absolute rates (within 5-fold; Table 2) and indeed affected the rates to a smaller degree than could have been expected based on assays comparing oxyazabenzotriazolides with 2-methylimidazolides. 24 For the RNA template, the imidazolides gave faster reactions than the OAt esters (by at most one order of magnitude). Stalling factors did not appear to be affected systematically by the identity of leaving group (Figure 2B; Supporting Figure S1B).…”

Section: Resultsmentioning

confidence: 99%

“…24 Mixing of equimolar quantities of the four OAt esters as previously described 19 gave solutions that were directly used for assays. Imidazolides of dNMPs were prepared via redox condensation using imidazole, 2,2′-dithiodipyridine and triphenylphosphine, as described for ribonucleotides by Lohrmann and Orgel.…”

Section: Methodsmentioning

confidence: 99%

“…Characterization data for the OAt esters were published previously 24 and ESI-MS are given in Supporting Figures S38-S39.…”

Section: Methodsmentioning

confidence: 99%

“…A sample of the supernatant was used for MALDI-TOF MS analysis, as previously described. 24 The estimated detection limit for minor extension products is ≤1 %.…”

Section: Methodsmentioning

confidence: 99%

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Cascade of Reduced Speed and Accuracy after Errors in Enzyme-Free Copying of Nucleic Acid Sequences

Leu¹,

Kervio

Obermayer³

et al. 2012

J. Am. Chem. Soc.

Self Cite

105

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Non-enzymatic, template-directed synthesis of nucleic acids is a paradigm for self-replicating systems. The evolutionary dynamics of such systems depend on several factors, including the mutation rates, relative replication rates, and sequence characteristics of mutant sequences. We measured the kinetics of correct and incorrect monomer insertion downstream of a primer-template mismatch (mutation), using a range of backbone structures (RNA, DNA, and LNA templates and RNA and DNA primers) and two types of 5′-activated nucleotides (oxyazabenzotriazolides and imidazolides, i.e., nucleoside 5’-phosphorimidazolides). Our study indicated that for all systems studied, an initial mismatch was likely to be followed by another error (54-75% of the time) and extension after a single mismatch was generally 10-100 times slower than extension without errors. If the mismatch was followed by a matched base pair, the extension rate recovered to nearly normal levels. Based on these data, we simulated nucleic acid replication in silico, which indicated that a primer suffering an initial error would lag behind properly extended counterparts due to a cascade of subsequent errors and kinetic stalling, with the typical mutational event consisting of several consecutive errors. Our study also included different sequence contexts, which suggest the presence of cooperativity among monomers affecting both absolute rate (by up to two orders of magnitude) and fidelity. The results suggest that molecular evolution in enzyme-free replication systems would be characterized by large ‘leaps’ through sequence space rather than isolated point mutations, perhaps enabling rapid exploration of diverse sequences. The findings may also be useful for designing self-replicating systems combining high fidelity with evolvability.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cascade of Reduced Speed and Accuracy after Errors in Enzyme-Free Copying of Nucleic Acid Sequences

Leu¹,

Kervio

Obermayer³

et al. 2012

J. Am. Chem. Soc.

Self Cite

105

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“…It was long considered that one major drawback of this type of polymerization was its error-proneness (close to 20% of misincorporation) [64], which would extremely reduce the applicability of this method for a nucleic acid of a length corresponding to known enzymatically-active RNA molecules. Irene Chen and co-workers [65,66] have however shown that the incorporation of a noncognate nucleobase at the 3′-end of a primer (terminated with 3′-NH 2 -ddNMP) reduces rates of a subsequent elongation by more than two orders of magnitude compared to those observed in the case of correct elongation.…”

Section: Rna Polymerization In Bulk Aqueous Mediamentioning

confidence: 99%

Taming Prebiotic Chemistry: The Role of Heterogeneous and Interfacial Catalysis in the Emergence of a Prebiotic Catalytic/Information Polymer System

Monnard

2016

Life

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Cellular life is based on interacting polymer networks that serve as catalysts, genetic information and structural molecules. The complexity of the DNA, RNA and protein biochemistry suggests that it must have been preceded by simpler systems. The RNA world hypothesis proposes RNA as the prime candidate for such a primal system. Even though this proposition has gained currency, its investigations have highlighted several challenges with respect to bulk aqueous media: (1) the synthesis of RNA monomers is difficult; (2) efficient pathways for monomer polymerization into functional RNAs and their subsequent, sequence-specific replication remain elusive; and (3) the evolution of the RNA function towards cellular metabolism in isolation is questionable in view of the chemical mixtures expected on the early Earth. This review will address the question of the possible roles of heterogeneous media and catalysis as drivers for the emergence of RNA-based polymer networks. We will show that this approach to non-enzymatic polymerizations of RNA from monomers and RNA evolution cannot only solve some issues encountered during reactions in bulk aqueous solutions, but may also explain the co-emergence of the various polymers indispensable for life in complex mixtures and their organization into primitive networks.

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Templated Self‐Replication in Biomimetic Systems

et al. 2019

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nanostructures and nanopatterns. [13][14][15] The specificity of the molecular recognitions between replicative units is concomitant with the encoding and transmission of information, with the degree of specificity determining the fidelty and capacity of information transfer against competing background interactions in noisy "chemical" environments. [16] However, the ability to replicate in this manner does not imply the ability to transmit additional heritable information-simple replicators can only replicate information related to recognition and template directed autocatalysis as this information is intrinsically linked to phenotype. In contrast to most artificial autocatalytic replicators, living systems (including viruses) store the key instructions for replication on an inheritable genetic system rather than in the native structures of the individual sub-components. This decoupling of phenotype and genotype enables heredity and open-ended evolution, the possibility of an indefinite increase in complexity, as evolutionary innovations aquired by natural selection are anchored in the genotype only. [17,18] The most prominent self-replicating molecules are nucleic acids, which form the fundamental basis for information storage and propagation in biological systems. The ability of polynucleic acids to store information is self-evident, and based upon the ability to form cognate Watson-Crick base pair interactions. Nucleic acid systems may have also formed the first genetic replicators in biology. The RNA world hypothesis postulates a stage in the origin of life in which RNA proliferated before the emergence of DNA and proteins. To do this, RNA must be able to carry out several key roles: information storage, catalysis, and (self-) replication. Multiple examples of catalytic RNAs (ribozymes) are known both from nature and in vitro selection experiments, [19][20][21][22] but there is no fossil evidence of an RNA capable of (self-)replication without the involvement of proteins.While minimal nucleic acid-only systems with replicationlike properties have been identified by directed evolution and engineering, limitations such as low activity, lack of generality, and poor information capacity limit their usefulness in bottomup synthetic biology, except in origin of life research. For the creation of more complex biological in vitro systems, RNA or DNA replication mechanisms involving more powerful protein enzymes are likely to be necessary. Even so, the achievement of self-sustained in vitro self-replication inspired by the central dogma of molecular biology is currently hampered by the requirement to encode and efficiently express the enormous A key characteristic of living systems is the storage and replication of information, and as such the development of self-replicating systems capable of heredity is of great importance to the fields of synthetic biology and origin of life research. In this review, the design and implementation of self-replicating systems in the context of bottom-up synthetic biology is discusse...

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Chemical Primer Extension: Efficiently Determining Single Nucleotides in DNA

Cited by 67 publications

References 28 publications

Cascade of Reduced Speed and Accuracy after Errors in Enzyme-Free Copying of Nucleic Acid Sequences

Cascade of Reduced Speed and Accuracy after Errors in Enzyme-Free Copying of Nucleic Acid Sequences

Taming Prebiotic Chemistry: The Role of Heterogeneous and Interfacial Catalysis in the Emergence of a Prebiotic Catalytic/Information Polymer System

Templated Self‐Replication in Biomimetic Systems

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