Mapping of Single-Base Differences between Two DNA Strands in a Single Molecule Using Holliday Junction Nanomechanics
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{"title"=>"Mapping of Single-Base Differences between Two DNA Strands in a Single Molecule Using Holliday Junction Nanomechanics", "type"=>"journal", "authors"=>[{"first_name"=>"Camille", "last_name"=>"Brème", "scopus_author_id"=>"13409237100"}, {"first_name"=>"François", "last_name"=>"Heslot", "scopus_author_id"=>"6602737081"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "doi"=>"10.1371/journal.pone.0055154", "pui"=>"368294459", "sgr"=>"84873509749", "pmid"=>"23393565", "scopus"=>"2-s2.0-84873509749"}, "id"=>"9f07ce79-0e21-3f5a-ad82-6e94e6d5d08b", "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.\\n\\nMETHODS: 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.\\n\\nMAIN 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.\\n\\nSIGNIFICANCE: 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.", "link"=>"http://www.mendeley.com/research/mapping-singlebase-differences-between-two-dna-strands-single-molecule-using-holliday-junction-nanom", "reader_count"=>9, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Student > Ph. D. Student"=>4, "Other"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>2, "Student > Ph. D. Student"=>4, "Other"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Mathematics"=>1, "Agricultural and Biological Sciences"=>3, "Physics and Astronomy"=>3, "Computer Science"=>1}, "reader_count_by_subdiscipline"=>{"Physics and Astronomy"=>{"Physics and Astronomy"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>3}, "Computer Science"=>{"Computer Science"=>1}, "Mathematics"=>{"Mathematics"=>1}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"France"=>1}, "group_count"=>1}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/494121"], "description"=>"<p>Fig. 1A: Conversion of a palindromic DNA molecule into a Holliday junction, in which the initial parental segments recombine to form heteroduplexes. If the recombining sequences differ by one base, two mismatches (one on each of the recombined arms) are formed. Fig. 1B: The molecule is nearly symmetric with respect to the center of the construct. In the opposite orientations, a first section of identical DNA segments is found near the center of each arm. This section is followed by an approximately 800-bp segment (oxa7 or oxa11) that exhibits a 5% difference between the sequences of the arms. The differing arm segments are followed by an identical DNA segment. The sequences of the regions that differ between the two arms are shown in the <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055154#pone.0055154.s002\" target=\"_blank\">Sequence Information S1</a>.</p>", "links"=>[], "tags"=>["holliday", "junction", "molecular"], "article_id"=>164640, "categories"=>["Biochemistry", "Biotechnology", "Biological Sciences", "Biophysics"], "users"=>["Camille Brème", "François Heslot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0055154.g001", "stats"=>{"downloads"=>2, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sketch_of_a_Holliday_junction_and_its_molecular_construction_/164640", "title"=>"Sketch of a Holliday junction and its molecular construction.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-05 01:17:20"}
  • {"files"=>["https://ndownloader.figshare.com/files/494364"], "description"=>"<p>The construct used contains a single base difference between the test sequences (see Methods). The experiments were performed with a HJ that was formed in Buff-A and manipulated in Buff-B. The initial force was approximately 0.2 to 0.3 pN, and the rotation was adjusted to induce the formation of plectonemes after HJ blockage (20 negative turns after the blockage). The force was then quickly raised to approximately 4 pN (“high stress” conditions), although the rotation was unchanged. A mismatch bypass was observed after a variable time. The number of events observed for a given waiting time interval was plotted as a function of the waiting time. The data shown in Fig. 6A and Fig. 6B were obtained at temperatures of 30°C and 37°C, respectively. The data were fit assuming an exponential decay (continuous line); the characteristic times obtained were approximately 19 and 3 seconds at 30°C and 37°C, respectively.</p>", "links"=>[], "tags"=>["bypassing", "blockage"], "article_id"=>164896, "categories"=>["Biochemistry", "Biotechnology", "Biological Sciences", "Biophysics"], "users"=>["Camille Brème", "François Heslot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0055154.g006", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Waiting_time_for_bypassing_a_blockage_under_8220_high_stress_8221_conditions_/164896", "title"=>"Waiting time for bypassing a blockage under “high stress” conditions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-05 01:21:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/494258"], "description"=>"<p>Experimental data in Buff-B (squares). Some of the data shown in Fig. 3 (obtained in Buff-A and the selected range of rotation) are superimposed (stars). The curve-segments U, X, V and Y were obtained sequentially in Buff-B starting from R = −486; these curves were obtained using the same molecule that was used to obtain the data shown in Fig. 3. Curve-segment U was obtained for R in the range of −486 to −480, curve-segment X was initiated by decreasing R from −479, curve-segment V was obtained for R in the range of −479 −472, and curve-segment Y was initiated by decreasing R from −471. Curve-segments U and V correspond to HJ migration, whereas curve-segments X and Y correspond to a blocked HJ (toward more negative values of R) with the formation of plectonemes and are designated later in the text as “plectoneme-curve-segments”.</p>", "links"=>[], "tags"=>["junction"], "article_id"=>164786, "categories"=>["Biochemistry", "Biotechnology", "Biological Sciences", "Biophysics"], "users"=>["Camille Brème", "François Heslot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0055154.g004", "stats"=>{"downloads"=>1, "page_views"=>40, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Holliday_junction_migration_or_blockage_/164786", "title"=>"Holliday junction migration or blockage.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-05 01:19:46"}
  • {"files"=>["https://ndownloader.figshare.com/files/494310"], "description"=>"<p>Fig. 5A: Experimental data of the relative height (with respect to the height at R = 0) as a function of the rotation R for the plectoneme-curve-segments obtained in Buff-B in the region R = −465 to R = −545 using the process described in the methods section. The vertical traction force is approximately 0.18 pN. The plectoneme-curve-segments were obtained sequentially, starting from the left-most plectoneme-curve-segment on the figure (see Methods). Fig. 5B: The rotations Ri for the different blockages were determined using a non-linear fitting procedure (see Methods). The figure shows a plot of the experimental data-derived Ri (Turns) for the blockages depicted in Fig. 3A as a function of the expected blockage rotation (Turns) deduced from the sequence. A linear fit y = A+x for the data was obtained using the Origin software (OriginLab), which yielded A = 0.05, R-value = 0.9998, and Standard Deviation (SD) = 0.45. To independently obtain error estimates on the blockage values, a bootstrap method was used (see Methods); the SD obtained by bootstrap method was in the range of 0.5 to 0.6 turns. Inset of Fig. 5B: Indices of the expected mismatches in the construct and the mismatches formed. The asterisk (*) indicates the mismatches (over 8 bases) that were not resolved from their nearest (*)-labeled neighbors; only one blocking event was recorded with an uncertainty in the attribution. The “middle” (*) mismatch was arbitrarily assumed to be the one detected.</p>", "links"=>[], "tags"=>["blockages"], "article_id"=>164836, "categories"=>["Biochemistry", "Biotechnology", "Biological Sciences", "Biophysics"], "users"=>["Camille Brème", "François Heslot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0055154.g005", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Experimental_blockages_and_data_treatment_/164836", "title"=>"Experimental blockages and data treatment.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-05 01:20:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/482488", "https://ndownloader.figshare.com/files/482492"], "description"=>"<div><h3>Objective</h3><p>The aim of this work is to demonstrate a novel single-molecule DNA sequence comparison assay that is purely based on DNA mechanics.</p> <h3>Methods</h3><p>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.</p> <h3>Main Results</h3><p>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.</p> <h3>Significance</h3><p>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.</p> </div>", "links"=>[], "tags"=>["single-base", "differences", "dna", "strands", "holliday", "junction", "nanomechanics"], "article_id"=>155833, "categories"=>["Biochemistry", "Biotechnology", "Biological Sciences", "Biophysics"], "users"=>["Camille Brème", "François Heslot"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0055154.s001", "https://dx.doi.org/10.1371/journal.pone.0055154.s002"], "stats"=>{"downloads"=>9, "page_views"=>45, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Mapping_of_Single_Base_Differences_between_Two_DNA_Strands_in_a_Single_Molecule_Using_Holliday_Junction_Nanomechanics__/155833", "title"=>"Mapping of Single-Base Differences between Two DNA Strands in a Single Molecule Using Holliday Junction Nanomechanics", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-02-05 01:37:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/494209"], "description"=>"<p>Fig. 3A: Different configurations (α, β, and γ) of the molecular construct: (α) no HJ, (β) HJ but no mismatch, and (γ) HJ with mismatches in the lateral arms. A Holliday junction that is formed by micromanipulation can be moved by manipulating the winding applied to the construct. This movement is associated with a progression of the recombination. Fig. 3B: Data on the relative height (with respect to the height at R = 0) as a function of the rotation R in buffer Buff-A. The vertical traction force is approximately 0.35 pN. The bell-shaped curve near R = 0 corresponds to the formation of plectonemes with no HJ present (Fig. 3A-α). The data points in regions β and γ correspond to HJ migration (Fig. 3A-β and 3A-γ).</p>", "links"=>[], "tags"=>["configuration", "holliday", "junction"], "article_id"=>164733, "categories"=>["Biochemistry", "Biotechnology", "Biological Sciences", "Biophysics"], "users"=>["Camille Brème", "François Heslot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0055154.g003", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Molecular_configuration_and_Holliday_junction_migration_or_blockage_/164733", "title"=>"Molecular configuration and Holliday junction migration or blockage.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-05 01:18:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/494166"], "description"=>"<p>The molecule is tethered between a glass surface and a paramagnetic bead. Multiple attachments at the extremities of the molecule ensure that the molecule is rotationally constrained. A pair of magnets, which are controlled above the sample by a motor, imposes the force and the rotation that is applied to the bead. Using video microscopy and concurrent image analysis of each video frame, the vertical and lateral positions of the tethered bead with respect to the surface of the sample are determined in real time. The extension of the molecule is deduced from the measurement of the average vertical position of the bead.</p>", "links"=>[], "tags"=>["biotechnology", "Computational biology", "biophysics", "Biochemistry"], "article_id"=>164686, "categories"=>["Biochemistry", "Biotechnology", "Biological Sciences", "Biophysics"], "users"=>["Camille Brème", "François Heslot"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0055154.g002", "stats"=>{"downloads"=>0, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Sketch_of_the_experimental_configuration_/164686", "title"=>"Sketch of the experimental configuration.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-05 01:18:06"}

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Relative Metric

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