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Mapping of single-base differences between two DNA strands in a single molecule using holliday junction nanomechanics.

ABSTRACT: OBJECTIVE: The aim of this work is to demonstrate a novel single-molecule DNA sequence comparison assay that is purely based on DNA mechanics. METHODS: A molecular construct that contained the two homologous but non-identical DNA sequences that were to be compared was prepared such that a four-way (Holliday) junction could be formed by the formation of heteroduplexes through the inter-recombination of the strands. Magnetic tweezers were used to manipulate the force and the winding applied to this construct for inducing both the formation and the migration of a Holliday junction. The end-to-end distance of the construct was measured as a function of the winding and was used to monitor the behavior of the Holliday junction in different regions of the intra-molecular recombination. MAIN RESULTS: In the appropriate buffer, the magnet rotation induces the migration of the Holliday junction in the regions where there is no sequence difference between the recombining sequences. In contrast, even a single-base difference between the recombining sequences leads to a long-lasting blockage of the migration in the same buffer; this effect was obtained when the junction was positioned near this locus (the site of the single-base difference) and forced toward the formation of heteroduplexes that comprise the locus. The migration blockages were detected through the identification of the formation of plectonemes. The detection of the presence of sequence differences and their respective mappings were obtained from the series of blockages that were detected. SIGNIFICANCE: This work presents a novel single-molecule sequence comparison assay that is based on the use of a Holliday junction as an ultra-sensitive nanomechanism; the mismatches act as blocking grains of sand in the Holliday "DNA gearbox". This approach will potentially have future applications in biotechnology.

SUBMITTER: Breme C

PROVIDER: S-EPMC3564857 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Mapping of single-base differences between two DNA strands in a single molecule using holliday junction nanomechanics.

Brème Camille C Heslot François F

PloS one 20130205 2

<h4>Objective</h4>The aim of this work is to demonstrate a novel single-molecule DNA sequence comparison assay that is purely based on DNA mechanics.<h4>Methods</h4>A molecular construct that contained the two homologous but non-identical DNA sequences that were to be compared was prepared such that a four-way (Holliday) junction could be formed by the formation of heteroduplexes through the inter-recombination of the strands. Magnetic tweezers were used to manipulate the force and the winding a ...[more]

PMID: 23393565

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Project description:The Holliday junction (HJ) is a central intermediate of various genetic processes, including homologous and site-specific DNA recombination and DNA replication. Elucidating the structure and dynamics of HJs provides the basis for understanding the molecular mechanisms of these genetic processes. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. These data led us to the conclusion that one hop can be more than 1 basepair (bp); moreover, we hypothesized that continuous runs over the entire sequence homology (5 bp) can occur. Direct measurements of the dependence of the fluorescence resonance energy transfer (FRET) value on the donor-acceptor (D-A) distance are required to justify this model and are the major goal of this article. To accomplish this goal, we performed single-molecule FRET experiments with a set of six immobile HJ molecules with varying numbers of bps between fluorescent dyes placed on opposite arms. The designs were made in such a way that the distances between the donor and acceptor were equal to the distances between the dyes formed upon 1-bp migration hops of a HJ having 10-bp homology. Using these designs, we confirmed our previous hypothesis that the migration of the junction can be measured with bp accuracy. Moreover, the FRET values determined for each acceptor-donor separation corresponded very well to the values for the steps on the FRET time trajectories, suggesting that each step corresponds to the migration of the branch at a defined depth. We used the dependence of the FRET value on the D-A distance to measure directly the size for each step on the FRET time trajectories. These data showed that one hop is not necessarily 1 bp. The junction is able to migrate over several bps, detected as one hop and confirming our model. The D-A distances extracted from the FRET properties of the immobile junctions formed the basis for modeling the HJ structures. The composite data fit a partially opened, side-by-side model with adjacent double-helical arms slightly kinked at the four-way junction and the junction as a whole adopting a global X-shaped form that mimics the coaxially stacked-X structure implicated in previous solution studies.

Dataset Information

Mapping of single-base differences between two DNA strands in a single molecule using holliday junction nanomechanics.

Publications

Mapping of single-base differences between two DNA strands in a single molecule using holliday junction nanomechanics.

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