Montserrat Terrazas

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.

RNA/aTNA Chimeras: RNAi Effects and Nucleases Resistance of Single and Double Stranded RNAs

The RNA interference pathway (RNAi) is a specific and powerful biological process, triggered by small non-coding RNA molecules and involved in gene expression regulation. In this work, we explored the possibility of increasing the biological stability of these RNA molecules by replacing their natural ribose ring with an acyclic l-threoninol backbone. In particular, this modification has been incorporated at certain positions of the oligonucleotide strands and its effects on the biological properties of the siRNA have been evaluated. In vitro cellular RNAi assays have demonstrated that the l-threoninol backbone is well tolerated by the RNAi machinery in both double and single-stranded fashion, with activities significantly higher than those evinced by the unmodified RNAs and comparable to the well-known phosphorothioate modification. Additionally, this modification conferred extremely strong resistance to serum and 3′/5′-exonucleases. In view of these results, we applied this modification to the knockdown of a therapeutically relevant human gene such as apolipoprotein B (ApoB). Further studies on the activation of the innate immune system showed that l-threoninol-modified RNAs are slightly less stimulatory than unmodified RNAs.

Synthesis and Properties of Oligonucleotides Carrying Isoquinoline Imidazo[1,2-a]azine Fluorescent Units

Oligonucleotides carrying novel fluorescent compounds with a dipolar isoquinoline imidazo[1,2-a]azine core were prepared. Analysis of the melting curves demonstrates that DNA duplexes carrying these fluorescent labels at their ends have a slight increase in DNA duplex stability. The UV absorption and fluorescent properties of the oligonucleotide conjugates were analyzed. The fluorescent label is sensitive to duplex formation, as cooperative melting curves are also observed at 366 nm and fluorescence has a large increase upon denaturation. Cell uptake studies allow observation of these fluorescently labeled oligonucleotides internalized into HeLa cells.

Functionalization of the 3′-Ends of DNA and RNA Strands with N-ethyl-N-coupled Nucleosides: A Promising Approach To Avoid 3′-Exonuclease-Catalyzed Hydrolysis of Therapeutic Oligonucleotides

The development of nucleic acid derivatives to generate novel medical treatments has become increasingly popular, but the high vulnerability of oligonucleotides to nucleases limits their practical use. We explored the possibility of increasing the stability against 3′‐exonucleases by replacing the two 3′‐terminal nucleotides by N‐ethyl‐N‐coupled nucleosides. Molecular dynamics simulations of 3′‐N‐ethyl‐N‐modified DNA:Klenow fragment complexes suggested that this kind of alteration has negative effects on the correct positioning of the adjacent scissile phosphodiester bond at the active site of the enzyme, and accordingly was expected to protect the oligonucleotide from degradation. We verified that these modifications conferred complete resistance to 3′‐exonucleases. Furthermore, cellular RNAi experiments with 3′‐N‐ethyl‐N‐modified siRNAs showed that these modifications were compatible with the RNAi machinery. Overall, our experimental and theoretical studies strongly suggest that these modified oligonucleotides could be valuable for therapeutic applications.

Synthesis and properties of small interfering RNA duplexes carrying 5-ethyluridine residues

Oligoribonucleotides carrying 5-ethyluridine units were prepared using solid-phase phosphoramidite chemistry. The introduction of the tert-butyldimethylsilyl group at the 2′-OH position proceeded in good yield and very high 2′-regioselectivity. RNA duplexes carrying 5-ethyluridine either at the sense or the guide strands display RNAi activity comparable to or slightly better than that of unmodified RNA duplexes. Gene suppression experiments using luciferase targets in SH-SY5Y cells show that the ethyl group is generally well accepted at all positions although a small decrease in RNA interference activity is observed when one 5-ethylU residue is incorporated in the 3′ overhangs.

Challenges and Opportunities for Oligonucleotide-Based Therapeutics by Antisense and RNA Interference Mechanisms

Oligonucleotide-based therapeutics may be one of the most promising approaches for the treatment of diseases. Although significant progress has been made in developing these agents as drugs, several hurdles remain to be overcome. One of the most promising approaches to overcome these difficulties is the preparation of modified oligonucleotides designed to increase cellular uptake and/or increase stability to nucleases. Herein, we report the developments done by our group in the synthesis of modified oligonucleotides directed to the generation of active compounds for gene inhibition. Specifically we will report the synthesis of novel nuclease-resistant oligonucleotides such as North bicyclo[3.1.0]hexane pseudosugars or N-coupled dinucleotide units. Also, the design of several siRNA conjugates carrying cell-penetrating peptides, lipids, intercalating agents, and carbohydrates will be described. Some of these novel derivatives show clear improvements in their biological and inhibitory properties.