Multivalent effects dictate the binding affinity of multiple ligands on one molecular entity to receptors. Integrins are receptors that mediate cell attachment through multivalent binding to peptide sequences within the extracellular matrix, and overexpression promotes the metastasis of some cancers. Multivalent display of integrin antagonists enhances their efficacy, but current scaffolds have limited ranges and precision for the display of ligands. Here we present an approach to study multivalent effects across wide ranges of ligand number, density, and three-dimensional arrangement. Using L-lysine γ-substituted peptide nucleic acids, the multivalent effects of an integrin antagonist were examined over a range of 1 to 45 ligands. The optimal construct improves the inhibitory activity of the antagonist by two orders of magnitude against the binding of melanoma cells to the extracellular matrix in both in vitro and in vivo models.

Molecular beacon strategies using PNA are currently restricted to fluorophore attachment to the ends of the PNA. We report the synthesis of PNA oligomers wherein fluorophores can be attached to the PNA backbone from novel gamma-lysine PNA monomers. Oligomers incorporating the modified PNA showed comparable thermal stability to the corresponding aegPNA oligomer with DNA. When the modified PNA oligomer was annealed with complementary DNA, the fluorescence intensity increased 4-fold over the unbound PNA. [structure: see text]

Numerous regulatory genes have G-rich regions that can potentially form quadruplex structures, possibly playing a role in transcription regulation. We studied a G-rich sequence in the BCL2 gene 176-bp upstream of the P1 promoter for G-quadruplex formation. Using circular dichroism (CD), thermal denaturation and dimethyl sulfate (DMS) footprinting, we found that a single-stranded oligonucleotide with the sequence of the BCL2 G-rich region forms a potassium-stabilized G-quadruplex. To study G-quadruplex formation in double-stranded DNA, the G-rich sequence of the BCL2 gene was inserted into plasmid DNA. We found that a G-quadruplex did not form in the insert at physiological conditions. To induce G-quadruplex formation, we used short peptide nucleic acids (PNAs) that bind to the complementary C-rich strand. We examined both short duplex-forming PNAs, complementary to the central part of the BCL2 gene, and triplex-forming bis-PNAs, complementary to sequences adjacent to the G-rich BCL2 region. Using a DMS protection assay, we demonstrated G-quadruplex formation within the G-rich sequence from the promoter region of the human BCL2 gene in plasmid DNA. Our results show that molecules binding the complementary C-strand facilitate G-quadruplex formation and introduce a new mode of PNA-mediated sequence-specific targeting.

On display: This model shows DNA (pink) annealed to a peptide nucleic acid (PNA; orange) containing several L‐lysine γ side chains (LKγ‐PNA) in the middle of the PNA oligomer. The LKγ‐PNA side chains project away from the nucleobase pairs (purple) and line the periphery of the duplex. This strategy can be used for the multifunctional display of various functional groups from the PNA residues without diminishing the binding affinity or selectivity to DNA.