Sequence-specific fluorescence detection of DNA by polyamide-thiazole orange conjugates.
Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation.
Fechter
Eric J
EJ
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Olenyuk
Bogdan
B
Dervan
Peter B
PB
D001483:Base Sequence; D052160:Benzothiazoles; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009691:Nucleic Acid Denaturation; D009757:Nylons / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics; D011804:Quinolines; D013844:Thiazoles / Q000737:chemistry*
D052160:Benzothiazoles; D005456:Fluorescent Dyes; D009757:Nylons; D009841:Oligonucleotides; D011804:Quinolines; D013844:Thiazoles; C051000:thiazole orange; D004247:DNA