Enzyme‐Free Ligation of 5′‐Phosphorylated Oligodeoxynucleotides in a <scp>DNA</scp> Nanostructure

Krämer, M.; Richert, Clemens

doi:10.1002/cbdv.201700315

Cited by 17 publications

(9 citation statements)

References 27 publications

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“…The same group had previously demonstrated that mononucleotides would undergo template‐directed polymerization when activated by EDC only if 1‐Ethyl or a similar organocatalyst was present in the reaction buffer [6a] . In contrast, this reagent appeared to inhibit the ligation of 5′‐phosphate DNA oligonucleotides within the context of a DNA origami structure [5a] . Given these mixed results, we decided to investigate the impact of 1‐Ethyl on our DNA ligation reactions in which the phosphate at the nick site is on the substrate 3′ terminus.…”

Section: Resultsmentioning

confidence: 99%

Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation**

et al. 2020

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Chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields can range from 30 to 95 % for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation by using a well-defined bimolecular test system and a water-soluble carbodiimide (EDC) as a phosphateactivating agent. Our results emphasize the interplay between template-substrate complex stability and the rates of the chemical steps of ligation, with 3' phosphate substrates providing yields near 100 % after 24 hours for particularly favorable reaction conditions. Ligation rates are also shown to be sensitive to the identity of the base pairs flanking a nick site, with as much as threefold variation. Finally, the observation that DNA substrates are modified by EDC at rates that can be comparable with ligation rates emphasizes the importance of considering side reactions when designing protocols to maximize ligation yields.

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Section: Resultsmentioning

confidence: 99%

Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation**

et al. 2020

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“…Ligation was a comparably simple reaction and the chemical details of its prebiotic implementation have been explored 10,[56][57][58][59][60] . It will be interesting to see how similar cooperative mechanisms could be established in base-by-base replication mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Cooperative Ligation Breaks Sequence Symmetry and Stabilizes Early Molecular Replication

Toyabe

Braun

2019

Phys. Rev. X

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Each living species carries a complex DNA sequence that determines their unique features and functionalities. It is generally assumed that life started from a random pool of oligonucleotides sequences, generated by a prebiotic polymerization of nucleotides. The mechanism that initially facilitated the emergence of sequences that code for the function of the first species from such a random pool of sequences remains unknown. It is a central problem of the origin of life. An interesting option would be a self-selection mechanism by spontaneous symmetry breaking. Initial concentration fluctuations of specific sequence motifs would have been amplified and outcompeted less abundant sequences, enhancing the signal to noise to replicate and select functional sequences. Here, we demonstrate with experimental and theoretical findings that templated ligation would provide such a self-selection. In templated ligation, two adjacent single sequences strands are chemically joined when a third complementary strand sequence brought them in close proximity. This simple mechanism was a likely side-product of a prebiotic polymerization chemistry once the strands reach the length to form double stranded species. As shown here, the ligation gave rise to a nonlinear replication process by the cooperative ligation of matching sequences which self-promoted their own elongation. This led to a cascade of enhanced template binding and faster ligation reactions. A requirement was the reshuffling of the strands by thermal cycling, enabled for example by microscale convection. By using a limited initial sequence space and performing long term ligations, we found that complementary sequences with an initially higher concentration prevailed over either non-complementary or less concentrated sequences. The latter died out by the molecular degradation that we simulated in the experiment by serial dilution. The experimental results are consistent with both explicit and abstract theory models that were generated considering the ligation rates determined experimentally. Previously, other nonlinear modes of replication such as Hypercycles have been discussed to overcome instabilities from first-order replication dynamics such as the error catastrophe and the dominance of structurally simple, but fast replicating sequences, known as the Spiegelman problem. Assuming that templated ligation was driven by the same chemical mechanism that generated prebiotic polymerization of oligonucleotides, the mechanism could function as a missing link between polymerization and the self-stabilized replication, offering a pathway to the autonomous emergence of Darwinian evolution for the origin of life.

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“…If so, the results may help to bridge the gap between oligomerization of monomers and copying with prebiotically plausible strand mixtures. Further, a methodology for ligating RNA strands would also be useful for practical applications, including the synthesis of circular RNA (39), labeled siRNA (40), modified mRNA (41) and functional nanostructures (42,43).…”

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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Enzyme‐Free Ligation of 5′‐Phosphorylated Oligodeoxynucleotides in a DNA Nanostructure

Cited by 17 publications

References 27 publications

Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation**

Towards Efficient Nonenzymatic DNA Ligation: Comparing Key Parameters for Maximizing Ligation Rates and Yields with Carbodiimide Activation**

Cooperative Ligation Breaks Sequence Symmetry and Stabilizes Early Molecular Replication

Enzyme-free ligation of dimers and trimers to RNA primers

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