To conjugate or not to conjugate? evaluating the potential use of cell-penetrating peptides for conjugation or complexation with oligonucleotides by surface plasmon resonance.

Oligonucleotides represent a class of molecules that exhibit remarkable therapeutic potential due to their unparalleled target specificity, yet they suffer from limited cellular uptake and lack of tissue selectivity. Extensive research is conducted with cell-penetrating peptides (CPPs) as delivery excipients due to their ability to translocate across cellular membranes and deliver cargo into cells. This study aims to investigate an innovative approach to rapidly, and with small amounts of compound, analyze and compare complexation of CPPs to oligonucleotides. The study applies surface plasmon resonance (SPR) to evaluate a comprehensive library of CPPs regarding their interaction with a double-stranded oligonucleotide to assess their potential as complexing molecules or whether the CPP should be chemically linked to the negatively charged oligonucleotide to ensure proximity. Specifically, a small interfering RNA (siRNA) was immobilized on a biotinylated chip, and solutions of 66 CPPs were subsequently injected to determine their binding stoichiometry with the siRNA. The most influential molecular properties of the CPPs were determined to be the positive charge-to-length ratio, the total number of positive charges, and the overall hydrophobicity of the CPP. These findings demonstrate the effectiveness and utility of SPR as a high throughput screening tool for selecting peptide/oligonucleotide pairs intended for complexation or conjugation.