Strand invasion by oligonucleotide--nuclease conjugates.
Conjugates consisting of staphylococcal nuclease crosslinked to oligonucleotides hybridize to supercoiled duplex DNA by Watson--Crick base-pairing. Here we describe this strand invasion. Affinity cleavage by these conjugates provides a probe for the local topology of the DNA duplex and is most efficient at a target DNA sequence known to form a cruciform. Additional supercoiling of the substrate DNA increases selective cleavage at other sequences. Hybridization of the conjugate to duplex DNA is temperature dependent and is stable over time. Affinity cleavage is not substantially inhibited by a 200-fold excess of the analogous unmodified oligonucleotide, demonstrating that hybridization of the unmodified oligonucleotide must be less favored and that the nuclease is involved in substrate binding. Surprisingly, affinity cleavage is also not effectively inhibited by complementary oligonucleotides unless they contain an extended 5'-sequence capable of separate interactions with the nuclease domain of the conjugate. These results suggest that the oligonucleotide-nuclease conjugate prefers to hybridize to target sequences which will allow interactions with both the oligonucleotide and the nuclease domains. Affinity cleavage by oligonucleotide-nuclease conjugates provides general insights for the design of oligonucleotides and their conjugates for strand invasion and affords a convenient competition assay for their hybridization.
Corey
D R
DR
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas 75235, USA.
Munoz-Medellin
D
D
Huang
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D015342:DNA Probes; D008836:Micrococcal Nuclease / Q000737:chemistry*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D013696:Temperature
D015342:DNA Probes; D009841:Oligonucleotides; D010455:Peptides; D004247:DNA; D008836:Micrococcal Nuclease