Labeling Small RNAs through Chemical Ligation at the 5′ Terminus: Enzyme‐Free or Combined with Enzymatic 3′‐Labeling

Vogel, Heike; Richert, Clemens

doi:10.1002/cbic.201200214

Cited by 18 publications

(16 citation statements)

References 89 publications

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“…14,16,20,33-35 The substituents of the imidazole ring have been found to influence the rate and regiospecificity of the polymerization reaction of RNA monomers, with 2-methylimidazole giving the greatest yield, 36 although 2-methylimidazole was shown to be a poorer catalyst for the extension of 2'-aminoterminal DNA primers than other imidazoles. 37,38 Imidazoles and 4-aminopyridines were also found to be favorable leaving groups for montmorillonite-promoted RNA polymerization compared to other possibilities. 39 However, the influence of the leaving group on mutation rates and stalling was previously unknown.…”

Section: Discussionmentioning

confidence: 93%

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.

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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: Discussionmentioning

confidence: 93%

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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“…We first tested phosphoramidate ligation on al inear model system.Arecent study on labeling siRNAs [19] provided the startingp oint for the search for reaction conditions. Figure 1 shows the three-strandd uplex employed.W ep repared the 3'aminoterminal DNA strand 1 using the method of Eisenhuth.…”

Section: Resultsmentioning

confidence: 99%

Phosphoramidate Ligation of Oligonucleotides in Nanoscale Structures

et al. 2016

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The folding of long DNA strands into designed nanostructures has evolved into an art. Being based on linear chains only, the resulting nanostructures cannot readily be transformed into covalently linked frameworks. Covalently linking strands in the context of folded DNA structures requires a robust method that avoids sterically demanding reagents or enzymes. Here we report chemical ligation of the 3'-amino termini of oligonucleotides and 5'-phosphorylated partner strands in templated reactions that produce phosphoramidate linkages. These reactions produce inter-nucleotide linkages that are isoelectronic and largely isosteric to phosphodiesters. Ligations were performed at three levels of complexity, including the extension of branched DNA hybrids and the ligation of six scaffold strands in a small origami.

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“…).Long, unmodified RNAs can be prepared by in vitro transcription from DNA templates. Functional groups can be incorporated by enzymatic or chemical ligation at the 5'end, [10][11][12] at the 3'-end, [13,14] or at random internal positions through the use of labeled nucleoside triphosphates during transcription. [4] Long RNAs labeled at defined internal positions are accessible in a multi-step semi-synthetic method by enzymatic ligation of short, labeled RNA strands.…”

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confidence: 99%