Dataset Information

Transient states during the annealing of mismatched and bulged oligonucleotides.

ABSTRACT: Oligonucleotide hybridization is crucial in various biological, prebiotic and nanotechnological processes, including gene regulation, non-enzymatic primer extension and DNA nanodevice assembly. Although extensive research has focused on the thermodynamics and kinetics of nucleic acid hybridization, the behavior of complex mixtures and the outcome of competition for target binding remain less well understood. In this study, we investigate the impact of mismatches and bulges in a 12 bp DNA or RNA duplex on its association (kon) and dissociation (koff) kinetics. We find that such defects have relatively small effects on the association kinetics, while the dissociation kinetics vary in a position-dependent manner by up to 6 orders of magnitude. Building upon this observation, we explored a competition scenario involving multiple oligonucleotides, and observed a transient low specificity of probe hybridization to fully versus partially complementary targets in solution. We characterize these long-lived metastable states and their evolution toward equilibrium, and show that sufficiently long-lived mis-paired duplexes can serve as substrates for prebiotically relevant chemical copying reactions. Our results suggest that transient low accuracy states may spontaneously emerge within all complex nucleic acid systems comprising a large enough number of competing strands, with potential repercussions for gene regulation in the realm of modern biology and the prebiotic preservation of genetic information.

SUBMITTER: Todisco M

PROVIDER: S-EPMC10954449 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Transient states during the annealing of mismatched and bulged oligonucleotides.

Todisco Marco M Ding Dian D Szostak Jack W JW

Nucleic acids research 20240301 5

Oligonucleotide hybridization is crucial in various biological, prebiotic and nanotechnological processes, including gene regulation, non-enzymatic primer extension and DNA nanodevice assembly. Although extensive research has focused on the thermodynamics and kinetics of nucleic acid hybridization, the behavior of complex mixtures and the outcome of competition for target binding remain less well understood. In this study, we investigate the impact of mismatches and bulges in a 12 bp DNA or RNA ...[more]

PMID: 38348869

Similar Datasets

Project description:BackgroundMany SNP discrimination strategies employ natural restriction endonucleases to discriminate between allelic states. However, SNPs are often not associated with a restriction site and therefore, a number of attempts have been made to generate sequence-adaptable restriction endonucleases. In this study, a simple, sequence-adaptable SNP discrimination mechanism between a 'wild-type' and 'mutant' template is demonstrated. This model differs from other artificial restriction endonuclease models as cis- rather than trans-orientated regions of single stranded DNA were generated and cleaved, and therefore, overcomes potential issues of either inefficient or non-specific binding when only a single variant is targeted.ResultsA series of mismatch 'bubbles' that spanned 0-5-bp surrounding a point mutation was generated and analysed for sensitivity to S1 nuclease. In this model, generation of oligonucleotide-mediated ssDNA mismatch 'bubbles' in the presence of S1 nuclease resulted in the selective degradation of the mutant template while maintaining wild-type template integrity. Increasing the size of the mismatch increased the rate of mutant sequence degradation, until a threshold above which discrimination was lost and the wild-type sequence was degraded. This level of fine discrimination was possible due to the development of a novel high-resolution melting curve assay to empirically determine changes in Tm (~5.0°C per base-pair mismatch) and to optimise annealing conditions (~18.38°C below Tm) of the mismatched oligonucleotide sets.ConclusionsThe in vitro 'cleavage bubble' model presented is sequence-adaptable as determined by the binding oligonucleotide, and hence, has the potential to be tailored to discriminate between any two or more SNPs. Furthermore, the demonstrated fluorometric assay has broad application potential, offering a rapid, sensitive and high-throughput means to determine Tm and annealing rates as an alternative to conventional hybridisation detection strategies.

Project description:Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3' end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. BIR still occurs about 10% as often when every 6th base is mismatched as with a perfectly matched donor. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, each with different spatial arrangements. Although 2 of the 6 arrangements we tested were nearly as efficient as the evenly-spaced reference, 4 were significantly less efficient. A donor with all 10 mismatches clustered at the 3' invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5' end. Our data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but mismatch position-specific effects are also important. We also addressed an apparent difference between in vitro and in vivo strand exchange assays, where in vitro studies had suggested that at a single contiguous stretch of 8 consecutive bases was needed to be paired for stable strand pairing, while in vivo assays using 108-bp substrates found significant recombination even when every 6th base was mismatched. Now, using substrates of either 90 or 108 nt-the latter being the size of the in vivo templates-we find that in vitro D-loop results are very similar to the in vivo results. However, there are still notable differences between in vivo and in vitro assays that are especially evident with unevenly-distributed mismatches. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3' end. Mismatch incorporation depends on the 3'→ 5' proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins, or from Rad1-Rad10 flap nuclease or the Mph1 helicase. Surprisingly, the probability of a mismatch 27 nt from the 3' end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase δ "chews back" the 3' end of the invading strand without any mismatch-dependent cues from the strand invasion structure. However, there appears to be an alternative way to incorporate a mismatch at the first base at the 3' end of the donor.

Dataset Information

Transient states during the annealing of mismatched and bulged oligonucleotides.

Publications

Transient states during the annealing of mismatched and bulged oligonucleotides.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets