The majority of previous approaches to incorporating labels into covalently closed circular DNA can be grouped into two categories. The first group is based on the initial approach of Zoller and Smith (29
) using a short synthetic oligonucleotide, complementary to the circular single-strand DNA, as a primer for DNA polymerase. A number of modifications and enhancements, such as using different polymerases, the addition of different ensembles of accessory proteins, and different reaction conditions, have been devised to reduce the limitations of low yields and/or displacement of the labeled oligonucleotide primer by polymerase elongating around the circle (30
). The second group consists of various methods for constructing gapped molecules, which were then filled in with a single oligonucleotide. Circular ssDNA was used as the source of one strand and an appropriate duplex DNA (made either by restriction endonuclease digestion or by PCR) as a source of the second strand (32
The method we have described here for incorporating a large number of synthetic oligonucleotides, any or all of which can carry a variety of different labels and/or DNA modifications, is well suited for analyses involving FRET. It is likely to be advantageous in many other types of analyses, as well. In the experiments reported here, we used three oligonucleotides to fill in a gap of 136
bp but we have also made supercoiled att
P substrates using five oligonucleotides to fill in a 263-bp gap with acceptable overall yields. In those cases where there are no constraints on the DNA sequences at one or both boundaries of the gap, the method can be simplified by using only one restriction enzyme to cut the ssDNA.
One of the features of the gapped molecule approach that is essential for our studies is the assurance that substrates are homogeneous and completely labeled. Our method is also well suited for experiments in which multiple labels must be positioned and/or repositioned throughout the course of a study. This method can be readily scaled up to produce large quantities of labeled supercoiled DNA for biochemical and structural studies, in which case it might be advantageous to replace the final gel electrophoresis purification of supercoiled substrate with CsCl2 density gradient centrifugation.
The FRET experiments reported here indicate the feasibility of this approach to studying the integrative recombination reaction, and, by extension, many other reactions in a wide range of systems that depend upon or utilize supercoiled DNA. The specific placement of labels and DNA modifications in supercoiled substrates should be particularly helpful in studies on the initiation and elongation steps of both replication and transcription, nucleosome positioning and many pathways of recombination (33–36
). Biophysical studies of DNA, including the internal dynamics of supercoiled DNA are also likely beneficiaries of such substrates (37
A potentially practical application of site-specifically modified supercoiled DNA is in the area of non-viral gene delivery into eukaryotic cells and non-viral gene therapy (2
). Among the modifications used to improve gene delivery to the nucleus are DNA–peptide conjugates, called nuclear localization sequences (NLS) (38
). However, most of the methods used thus far do not allow controlled addition of NLS peptide molecules to supercoiled DNA (39
). This could be important, since a single NLS attached to a DNA molecule is sufficient for nuclear entry, while several peptides might inhibit entry (40
). Additionally, too many chemical modifications of the delivery DNA (and/or in the wrong place) could inhibit transcription (41
). [For a recent review see ref. (42
According to the Förster theory (43
), the efficiency of resonance energy transfer
is related to the distance between the donor and acceptor,
, by the equation:
is the distance between the donor and acceptor that gives 50% FRET (the Förster distance). There are many factors complicating accurate determination of
); however, in the experiments reported here, the donor and acceptor dyes are in the same immediate environment (with respect to DNA sequence and protein neighbors) such that, whatever the precise value of ,
it should be the same in both the Holliday junction and the recombinant product. In other words, the ratio of the two FRET energies will be a measure of the ratio of the physical distances:
. The values ( and text above) for
(from the Holliday junction complex), and
(from the recombinant product complex), are 0.23 and 0.18, respectively, yielding 0.95 for the approximate ratio of the distances observed in the Holliday junction and product complexes.
These data strongly indicate that the portion of the higher-order structure revealed by FRET in the supercoiled Holliday junction intermediate is preserved in the linear recombination product. This had been an open question and the results raise additional questions about how many other features of the Holliday junction intermediate are preserved in the products and which of these preserved and/or altered features contributes to the directionality and regulation of the recombination reaction. Fortunately, the experiments reported here also open the door to answering many of the questions that have been raised in regard to the integrative reaction. It is not unreasonable to expect these methods, which are efficient, robust and readily scaled up, will also be useful in other reactions and systems involving supercoiled DNA.