Sandra M. Ocampo

Effect of North Bicyclo(3.1.0)hexane 2'-Deoxy- pseudosugars on RNA Interference: A Novel Class of siRNA Modification

North bicyclo methanocarba thymidine (TN) nucleosides were substituted into siRNAs to investigate the effect of bicyclo[3.1.0]hexane 2′‐deoxy‐pseudosugars on RNA interference activity. Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery. We studied the effect of the TN modification in a screen involving residue‐specific changes in an siRNA targeting Renilla luciferase and we applied the most effective pattern of modification to the knockdown of murine tumor necrosis factor (TNF‐α). We also showed that incorporation of TN units into siRNA duplexes increased their thermal stabilities, substantially enhanced serum stabilities, and decreased innate immunostimulation. Comparative RNAi studies involving the TN substitution and locked nucleic acids (LNAs) showed that the gene‐silencing activities of TN‐modified siRNAs were comparable to those obtained with the LNA modification. An advantage of the North 2′‐deoxy‐methanocarba modification is that it may be explored further in the future by changing the 2′‐position. The results from these studies suggest that this modification might be valuable for the development of siRNAs for therapeutic applications.

Synthesis and in vitro inhibition properties of siRNA conjugates carrying glucose and galactose with different presentations

Oligoribonucleotide conjugates and the corresponding siRNA duplexes against tumor necrosis factor carrying one, two, or four glucose and galactose residues at the 5′-end have been prepared using phosphoramidite chemistry. Carbohydrate-modified siRNA duplexes have similar inhibitory properties than unmodified RNA duplexes in HeLa cells transfected with oligofectamine. When HeLa cells were treated with siRNA carrying one, two, or four glucose residues without oligofectamine, no inhibition was observed. The inhibitory properties of siRNA carrying galactose residues without transfecting agent were tested on HuH-7 cells that have abundant asialoglycoprotein receptors. In these cells siRNA carrying galactose residues have slight anti-TNF inhibitory properties (25% in the best case) that are eliminated if the receptors are blocked with a competitor. These results demonstrate receptor-mediated uptake of siRNA carrying galactose residues, although the efficacy of the process is not enough for efficient RNA interference experiments.

Stepwise synthesis of RNA conjugates carrying peptide sequences for RNA interference studies

Oligoribonucleotide conjugates carrying nuclear localization peptide sequences at the 3′-end were prepared stepwise on a single support. The siRNA duplex carrying the nuclear localization peptide sequence at the 3′-end of the passenger strand has similar inhibitory properties as those of unmodified or cholesterol-modified RNA duplexes.

Synthesis of lipid-oligonucleotide conjugates for RNA interference studies.

The synthesis of RNA molecules carrying lipids at their 3′‐termini and 5′‐termini is reported. These conjugates were fully characterized by MALDI‐TOF mass spectrometry and HPLC chromatography. The ability of these conjugates to silence gene expression was evaluated in the inhibition of the tumor necrosis factor. All the lipidsiRNA derivatives were compatible with RNA interference machinery if transfected with oligofectamine. In the absence of a transfection agent, some lipidsiRNA derivatives can exert a slight reduction of gene expression.

Synthesis and in vitro Inhibition Properties of siRNA Conjugates Carrying Acridine and Quindoline Moieties

The synthesis of RNA molecules carrying acridine or quindoline residues at their 3′‐ and 5′‐termini is reported. These conjugates are fully characterized by MALDI‐TOF mass spectrometry. Modified siRNA duplexes carrying acridine or quindoline moieties were evaluated for inhibition of the tumor necrosis factor. The conjugates showed inhibitory properties similar to those of unmodified RNA duplexes in HeLa cells transfected with oligofectamine. The fluorescent properties of acridine derivatives allow direct observation of the cytoplasmatic distribution of modified siRNA inside the cells.

Branched RNA: A New Architecture for RNA Interference

Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems.

New developments in the synthesis of oligonucleotide-peptide conjugates

The stability of oligodeoxynucleotides to trifluoroacetic acid is studied. Pyrimidine oligonucleotides were stable in the conditions used for the removal of t-butyl groups. Oligonucleotide-3′-peptide conjugates carrying pyrimidine oligonucleotides are prepared stepwise using peptide-supports and Fmoc, t-butyl strategy. Using this strategy we have prepared an oligonucleotide-peptide conjugate containing as peptide the leucine-rich fragment of FOS, a transcription factor involved in many important cellular processes. This conjugate has a long peptide sequence with a large number of trifunctional amino acids.