Detection of single DNA molecules by multicolor quantum-dot end-labeling

Observation of DNA–protein interactions by single molecule fluorescence microscopy is usually performed by using fluorescent DNA binding agents. However, such dyes have been shown to induce cleavage of the DNA molecule and perturb its interactions with proteins. A new method for the detection of surface-attached DNA molecules by fluorescence microscopy is introduced in this paper. Biotin- and/or digoxigenin-modified DNA fragments are covalently linked at both extremities of a DNA molecule via sequence-specific hybridization and ligation. After the modified DNA molecules have been stretched on a glass surface, their ends are visualized by multicolor fluorescence microscopy using conjugated quantum dots (QD). We demonstrate that under carefully selected conditions, the position and orientation of individual DNA molecules can be inferred with good efficiency from the QD fluorescence signals alone. This is achieved by selecting QD pairs that have the distance and direction expected for the combed DNA molecules. Direct observation of single DNA molecules in the absence of DNA staining agent opens new possibilities in the fundamental study of DNA–protein interactions. This work also documents new possibilities regarding the use of QD for nucleic acid detection and analysis

In vitro selection of oligonucleotides that bind double-stranded DNA in the presence of triplex-stabilizing agents

A SELEX approach has been developed in order to select oligonucleotides that bind double-stranded DNA in the presence of a triplex-stabilizing agent, and was applied to a target sequence containing an oligopurine–oligopyrimidine stretch. After only seven rounds of selection, the process led to the identification of oligonucleotides that were able to form triple helices within the antiparallel motif. Inspection of the selected sequences revealed that, contrary to GC base pair which were always recognized by guanines, recognition of AT base pair could be achieved by either adenine or thymine, depending on the sequence context. While thymines are strongly preferred for several positions, some others can accommodate the presence of adenines. These results contribute to set the rules for designing oligonucleotides that form stable triple helices in the presence of triplex-stabilizing agents at physiological pH. They set the basis for further experiments regarding extension of potential target sequences for triple-helix formation or recognition of ligand–DNA complexes

Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level

Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem–loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 ± 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules