Interactions of two large antiviral polyamides with the long control region of HPV16.

PA1 and PA25 are large hairpin polyamides that are effective in nearly eliminating HPV16 episomes (DNA) in cell culture, and PA25 has broad spectrum activity against three cancer-causing forms of HPV (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are the interactions of these PAs with sequences in the long control region (LCR) of HPV16 (7348-122). Using an FeEDTA conjugate of PA1 (designed to recognize 5'-W2GW7-3'; W = A or T), 34 affinity cleavage (AC) patterns were detected for this fragment. These sites can be rationalized with sequences featuring perfect, single, double, triple and quadruple mismatches. Quantitative DNase I footprinting analysis indicates that perfect sites bind PA1 with Kds between 0.7 and 2.2 nM. Kds for single, double, triple and quadruple mismatch sites range from 1-3 nM-20 nM. Using AC and EDTA conjugates, we report that unlike smaller 8-ring hairpin PAs, introduction of a chiral turn in this large polyamide has no effect on binding orientation (forward vs. reverse). Despite its design to recognize 5'-W2GW5GW4-3' via two Im residues, a motif not represented in this HPV sequence, a PA25-EDTA conjugate yielded 31 affinity cleavage sites on the region. Low nM Kds for PA25 without EDTA indicates a high tolerance for triple and quadruple mismatches. While there is extensive coverage of the sequence examined, AC cleavage patterns for the two PAs show discrete binding events and do not overlap significantly. This indicates that within the context of A/T rich sequences, these PAs do not recognize a simple shared sequence-related feature of the DNA. These insights continue to inform the complex nature of large hairpin PA-DNA interactions and antiviral behavior.

Binding studies of a large antiviral polyamide to a natural HPV sequence.

PA1 is a large hairpin polyamide (dImPyPy-ß-PyPyPy-γ-PyPy-ß-PyPyPyPy-ß-Ta; Py = pyrrole, Im = imidazole, ß = beta alanine) that targets the sequence 5'-WWGWWWWWWW-3' (W = A or T) and is effective in eliminating HPV16 in cell culture (Edwards, T. G., Koeller, K. J., Slomczynska, U., Fok, K., Helmus, M., Bashkin, J. K., Fisher, C., Antiviral Res. 91 (2011) 177-186). Described here are its DNA binding properties toward a natural DNA, a 523 bp portion of HPV16 (2150-2672) containing three predicted perfect match sites. Strategies for obtaining binding data on large fragments using capillary electrophoresis are also described. Using an Fe EDTA conjugate of PA1, 19 affinity cleavage (AC) patterns were detected for this fragment. In many cases, there are multiple possible binding sequences (perfect, single and double mismatch sites) consistent with the AC data. Quantitative DNase I footprinting analysis indicates that perfect and most single mismatch sites bind PA1 with Kds between 0.7 and 4 nM, indicating excellent tolerance for the latter. Double mismatch sites exhibit Kds between 12 and 62 nM. A large fraction of the accessible sequence is susceptible to PA1 binding, much larger than predicted based on the literature of polyamide-DNA recognition rules.