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

Area of Science:

• Molecular biology
• Antiviral drug development
• DNA-polypeptide interactions

Background:

• Large hairpin polyamides (PAs) like PA1 and PA25 demonstrate significant efficacy in reducing HPV16 episomes in cell cultures.
• PA25 exhibits broad-spectrum activity against multiple cancer-causing HPV types.
• Understanding the precise DNA binding mechanisms of these PAs is crucial for optimizing their therapeutic potential.

Purpose of the Study:

• To investigate the DNA binding interactions of large hairpin polyamides PA1 and PA25 with the long control region (LCR) of HPV16.
• To characterize the binding affinity and sequence specificity of these polyamides, including their tolerance to DNA mismatches.
• To elucidate the impact of structural modifications, such as chiral turns, on polyamide binding orientation.

Main Methods:

• Affinity cleavage (AC) using FeEDTA conjugates of PA1 and PA25 to map DNA binding sites on HPV16 LCR.
• Quantitative DNase I footprinting to determine binding affinities (Kd values) for various DNA sequences.
• Analysis of binding orientation (forward vs. reverse) in relation to polyamide structure.

Main Results:

• PA1 and PA25 identified numerous binding sites within the HPV16 LCR, with varying degrees of sequence complementarity.
• PA1 exhibited high binding affinities (low nM Kd) for perfect and mismatched DNA sequences.
• PA25 demonstrated significant tolerance for triple and quadruple DNA mismatches, with low nM Kd values.
• Chiral turn introduction in large polyamides did not alter binding orientation, unlike in smaller PAs.
• Distinct, non-overlapping binding patterns for PA1 and PA25 suggest they do not share a simple, common sequence recognition feature in A/T-rich regions.

Conclusions:

• Large hairpin polyamides PA1 and PA25 bind to HPV16 LCR with high affinity and remarkable tolerance for DNA sequence variations.
• The binding characteristics suggest a complex recognition mechanism beyond simple sequence complementarity, particularly in A/T-rich DNA.
• These findings provide valuable insights into large polyamide-DNA interactions, supporting their development as novel antiviral agents against HPV infections.