Single molecule detection of direct, homologous, DNA/DNA pairing

Abstract

Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the "default" option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here.

Fig. 1.

Pairing of homologous DNAs using the parallel single molecule, magnetic tweezers-based assay. (A) Experimental approach. The black lines represent dsDNA molecules and the two samples are distinguished by red diamonds or green circles attached to their ends corresponding to Dig- and biotin labels, respectively. The dsDNA samples after incubation are mixed with superparamagnetic beads (gray circles) and incubated inside a capillary. (B) Image showing a black region that corresponds to the capillary. The distance between the inner capillary surface and the center of the bead is approximately 14 μm, corresponding to a DNA extension of approximately 12.5 μm. The asterisks show beads that are out of focus because their corresponding molecules are tethered to positions other than the edge of the (round) capillary that is in focus. Out-of-focus beads are not counted in the assay.

Fig. 2.

Pairing of Dig-labeled λ DNA and biotin labeled λ DNA. (A) Number of tethered beads vs. time in 87 μg/mL λ DNA in PBS and incubated at 37 °C. (B) Number of tethered beads vs. square of the DNA concentration.

Fig. 3.

Effect of sequence on DNA pairing. Distribution of extensions for about 100 beads at 0.4 pN for Dig-labeled λ phage dsDNA incubated with biotinylated molecules subregions from λ phage: 5-kb fragment bp #21613 (blue) and 5 kb fragment presenting tails (magenta outline); 5-kb fragment bp #11302 (green); 5 kb fragment bp #116 (purple); biotinylated λ phage (yellow). λ phage molecules with both labels are shown for comparison (gray outline). The schematic representations are shown to the right of each panel.

Fig. 4.

Intermolecular pairing between 6 μg/mL biotinylated 5-kb fragments and 60 μg/mL Dig-labeled λ phage in the presence of crowding agent and nonspecific competitors. (A) Pairing of Dig-labeled λ DNA with 5 kb subregion bp #21613 with and without 15% p/v PEG 8000. (B) Pairing of Dig-labeled λ DNA with 5 kb subregion bp #21613 in the presence of high concentration of fish sperm DNA (orange), pcDNA3.1 (purple) and control without competitor (blue).

Fig. 5.

Effect of temperature and salt on the DNA-DNA interaction. (A) Number of tethered beads vs. temperature for biotinylated λ phage and Dig-labeled λ phage, 83 μg/mL in PBS incubated during several time intervals at 37 °C; the values on the y axis correspond to an incubation time of 24 h. The data for other incubation times has been normalized so that all data sets have the same value at 37 °C. (B) Number of tethered beads vs. salt concentration for 60 μg/mL biotinylated λ phage and 60 μg/mL Dig-labeled λ phage incubated for 1 h at 37 °C in phosphate buffer 10 mM: NaCl (blue) and KCl (red).