Cationic siRNAs Provide Carrier-Free Gene Silencing in Animal Cells

siRNA-mediated gene silencing requires intracellular delivery of the nucleic acid. We have developed a carrierless molecular approach that follows the same cell entry route as cationic supramolecular complexes, yet should avoid the extracellular barriers encountered by nanoparticles. Cationic oligospermine−oligonucleotide conjugates (ZNAs, for Zip Nucleic Acids) were synthesized stepwise on an oligonucleotide synthesizer using a DMT-spermine phosphoramidite derivative. They were shown to enter cells and have access to the cytoplasm, provided their formal charge ratio N/P was >1.5. Cationic siRNAs that fulfilled this condition were shown to achieve selective inhibition of luciferase gene expression in the submicromolar concentration range in constitutively luciferase-expressing cells

Oligonucleotide−Oligospermine Conjugates (Zip Nucleic Acids): A Convenient Means of Finely Tuning Hybridization Temperatures

Synthesis of oligonucleotide probes and control of their hybridization temperature are key aspects of polymerase chain reaction (PCR)-based detection of genetic sequences. A straightforward means to approach the last goal is to decrease the repulsion between the polyanionic probe and target strands. To this end, we have developed a versatile automated synthesis of oligonucleotide−oligospermine derivatives that gave fast access to a large variety of compounds. Plots of their hybridization temperatures Tm vs overall charge provided a measure of the impact of interstrand phosphate repulsion (and of spermine-mediated attraction) on the main driving force of duplex formation, i.e., base pairing. It showed that stabilization brought about by excess cationic charges can be of larger absolute magnitude than interstrand repulsion, even in high salt media. Base sequence and conjugation site (3′ or 5′) hardly influenced the effect of spermine on Tm. In typical PCR probe conditions, the Tm increased linearly with the number of grafted spermines (e.g., 6.2 °C per spermine for a decanucleotide probe). The large data set of Tm vs number of spermines and oligonucleotide length allowed us to empirically derive a simple mathematical relation that is accurately predicting the Tm of any oligonucleotide−oligospermine derivative. Zip nucleic acids (ZNA) are thus providing an interesting alternative to locked nucleic acids (LNA) or minor groove binders (MGB) for raising the stability of 8−12-mer oligonucleotides up to ca. 70 °C, the level required for quantitative PCR experiments