Development of antisense alpha-helical peptide nucleic acids

A new class of nucleic acid surrogate, namely alpha helical peptide nucleic acid (αPNA), was proposed for antisense therapeutics. This proposed surrogate is composed of a peptide backbone leading to a defined secondary structure (α-helical motif) upon binding to their nucleic acid targets. Nucleobases are attached to sidechains of amino acids at regular intervals allowing for sequence specific Watson-Crick base pairing. Because of its unique structure, αPNA is expected to overcome some of the major problems that are associated with current antisense therapeutics.; The synthetic route to the pyrimidine nucleoamino acid building blocks was established as well as an improved solid phase strategy for incorporating cytosine nucleoamino acids. Two classes of αPNAs were designed and synthesized each differing in overall charge of the peptide backbone. Preliminary biophysical studies of these αPNAs with ssDNAs revealed that αPNAs could bind to ssDNAs strongly and sequence specifically. The orientation of the αPNAs (parallel versus antiparallel) with respect to the ssDNAs affected the affinity and the nature of the binding. NMR experiments (1D NMR, NOESY) provided direct evidence of Watson-Crick base pairing in monomeric αPNA and ssDNA complexes.; Modifications of the αPNA were then explored as a means of enhancing binding affinity and specificity. Strategically placed aliphatic groups on the N terminus of αPNAs could be used to modulate the affinity and orientational specificity of αPNA's complexation to ssDNA. Different αPNA backbones that vary the number of amino acids between the nucleoamino acid residues as well as enantiomeric αPNAs that incorporate “unnatural” D-amino acids affected the binding properties of the complexes. To evaluate the hybridization properties of the modified αPNAs with nucleic acids in a streamlined fashion, a fluorescence based hybridization assay was developed to screen αPNA libraries.; Finally αPNAs, especially those with N-terminal modified by hydrophobic groups were found to bind ssRNA with high affinity and sequence specificity. Also, αPNAs are stable in human serum and can permeate the cell membrane within hours. These studies provide the significant foundation for αPNA to become an effective antisense candidate.