Fluorescent marker for direct detection of specific dsDNA sequences

Abstract

We have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. The marker consists of a biotinylated enzyme, attached through the biotin-avidin interaction to a fluorescent nanosphere. Control over biotin position with respect to the enzyme's binding pocket is achieved by biotinylating the mutant EcoRI at the mutation site. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. Marker-laden DNA is then fluorescently stained and stretched on polylysine-coated glass slides so that the positions of the bound markers along individual DNA molecules can be measured. We demonstrate the marker's ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods.

Figure 1

(a) Schematic representation of the mutant EcoRI marker bound to stained ds DNA. (b) X-ray structure of the wild-type EcoRI specific complex. Coordinates are determined from a highly refined version of the protein data base (PDB) entry 1CKQ., DNA is indicated in magenta, residues C218 (yellow) and K249C (red) are shown as spheres. The K249C mutation was modeled into the wild-type structure using Pymol. C218 and K249C are located in the main domain, away from the DNA-binding cleft.

Figure 2

Bulk confirmation of mutant enzyme activity and biotinylation by gel-shift assay. Lanes 1 and 2: 391-bp oligo containing a single copy of EcoRI’s target sequence GAATTC, with and without streptavidin. Lanes 3 and 4: Bound mutant EcoRI (40 nM) decreases the oligo’s mobility through the gel, causing a band shift. Lane 5: Biotinylation does not affect enzyme binding. Lane 6: Streptavidin-EcoRI complex binds to oligo, causing a larger band shift.

Figure 3

Bulk confirmation of biotinylated mutant enzyme specificity using gel-shift assay. Mutant and biotinylated mutant enzymes cause shifts of the 391-bp target oligo (Lanes 3 and 5) but not of a 112-bp oligo lacking the recognition sequence placed in competition for binding (Lanes 4 and 6). Neither mutant nor biotinylated mutant cause a shift in the 376-bp star site oligo (Lanes 9 and 10), which contains the sequence 5′ GAATCC 3′, a single bp substitution from the EcoRI target.

Figure 4

Representative fluorescence images of marker-tagged λ-DNA, stretched and immobilized on slides, with the corresponding fluorescence intensity profiles along the DNA backbone; bead positions are clearly evident as peaks in the profiles. Dashed vertical lines indicate expected EcoRI target positions 1-5 on fully stretched λ-DNA. Scale bar is 5 μm.

Figure 5

Histogram of measured binding positions for 204 markers bound to slide-immobilized λ-DNA molecules. The solid line is a Gaussian fit to the data. Dashed vertical lines indicate expected EcoRI target sites 1-5 on fully stretched λ-DNA.