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.

Paradoxical effects of DNA binding polyamides on HTLV-1 transcription.

Human T-cell leukemia virus type-1 (HTLV-1) depends on the virally encoded transcription factor Tax for efficient viral replication and gene expression. In a complex with CREB, Tax contacts the minor groove of the promoter DNA at guanine and cytosine rich sequences that flank three of the off-consensus cyclic-AMP response elements (CREs). In this study, we used six Tax-directed pyrrole-imidazole polyamides specifically designed to block Tax binding to DNA at each GC sequence of the three viral CREs. We found that four of these polyamides disrupt binding of the Tax/CREB complex in vitro, and that these same molecules also inhibit Tax-mediated transcription in vitro on chromatin-assembled templates. However, of these four Tax/CREB-specific polyamides, only one polyamide appears to be uniquely Tax specific. We show that polyamides can enter the nuclei of HTLV-1 infected T-cells, and two of the four polyamides down-regulated virion production in these cells. Together, these data illustrate the importance of studying polyamide inhibition of gene expression in vitro and in vivo, as the function of the polyamides in living cells is not fully understood. Finally, our data indicates that targeted disruption of the Tax/CREB complex, or other complexes which assemble on the HTLV-1 promoter, may provide a novel approach for inhibiting viral replication in vivo.

Allosteric inhibition of protein--DNA complexes by polyamide--intercalator conjugates.

The sequence-specific inhibition of essential protein-DNA contacts in the promoter of a gene is a central issue for the regulation of gene expression by chemical methods. Hairpin polyamides have been shown to inhibit protein-DNA complexes in some but not all cases. For example, polyamides co-occupy the same DNA sequence in the minor groove in the presence of major-groove binding bZip proteins. Four hairpin polyamide-acridine conjugates were synthesized and shown to bind the minor groove of DNA with high affinity in a sequence-specific manner. The polyamide-acridine conjugates were shown to unwind DNA (phi = 14-15 degrees), evidence for intercalation by the acridine moiety. Importantly, the polyamide-intercalator conjugates, which combine sequence-specific groove binding with proximal local unwinding, inhibit major-groove DNA binding by the GCN4 bZip protein. This class of DNA binding molecules creates a sequence-specific allosteric change in DNA structure and has the potential to be a general inhibitor of transcription factor binding independent of the specific protein-DNA structure.