Site-specific labeling of supercoiled DNA at the A+T rich sequences.
Progress in structural biology studies of supercoiled DNA and its complexes with regulatory proteins depends on the availability of reliable and routine procedures for site-specific labeling of circular molecules. For this, we made use of oligonucleotide uptake by plasmid DNA under negative superhelical tension. Subsequent circularization of the oligonucleotide label facilitated by an oligonucleotide scaffold results in its threading between the two strands of duplex DNA. Several lines of evidence, including direct AFM mapping of the label, show that the circular oligonucleotide is stably localized at its target, an A+T rich region. The specific binding mode when the oligonucleotide threads the double helix results in a DNA kink that tends to occupy an apical position in a plectonemically wound supercoiled DNA, similar to the positioning of an A-tract bend. Site-specific labels may allow visualization techniques, such as electron and atomic force microscopies, to reliably map protein binding sites, identify local alternative structures in supercoiled DNA, and monitor structural dynamics of DNA molecules in real time. Site-specific oligonucleotide reactions with DNA may also have application in biotechnology and gene therapy.
Potaman
Vladimir N
VN
Center for Genome Research, Institute of Biosciences and Technology, Texas A&M University System Health Sciences Center, Houston, Texas 77030-3303, USA. vpotaman@ibt.tamu.edu
Lushnikov
Alexander Y
AY
Sinden
Richard R
RR
Lyubchenko
Yuri L
YL
D000225:Adenine / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015342:DNA Probes / Q000138:chemical synthesis / Q000378:metabolism* / Q000648:ultrastructure; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D018900:Dinucleotide Repeats; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013941:Thymine / Q000378:metabolism*
D015342:DNA Probes; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D009838:Oligodeoxyribonucleotides; D000225:Adenine; D013941:Thymine