Stimuli-responsive oligonucleotides in prodrug-based approaches for gene silencing

Oligonucleotides (ONs) have been envisaged for therapeutic applications for more than thirty years. However, their broad use requires overcoming several hurdles such as instability in biological fluids, low cell penetration, limited tissue distribution, and off-target effects. With this aim, many chemical modifications have been introduced into ONs definitively as a means of modifying and better improving their properties as gene silencing agents and some of them have been successful. Moreover, in the search for an alternative way to make efficient ON-based drugs, the general concept of prodrugs was applied to the oligonucleotide field. A prodrug is defined as a compound that undergoes transformations in vivo to yield the parent active drug under different stimuli. The interest in stimuli-responsive ONs for gene silencing functions has been notable in recent years. The ON prodrug strategies usually help to overcome limitations of natural ONs due to their low metabolic stability and poor delivery. Nevertheless, compared to permanent ON modifications, transient modifications in prodrugs offer the opportunity to regulate ON activity as a function of stimuli acting as switches. Generally, the ON prodrug is not active until it is triggered to release an unmodified ON. However, as it will be described in some examples, the opposite effect can be sought.This review examines ON modifications in response to various stimuli. These stimuli may be internal or external to the cell, chemical (glutathione), biochemical (enzymes), or physical (heat, light). For each stimulus, the discussion has been separated into sections corresponding to the site of the modification in the nucleotide: the internucleosidic phosphate, the nucleobase, the sugar or the extremities of ONs. Moreover, the review provides a current and detailed account of stimuli-responsive ONs with the main goal of gene silencing. However, for some stimuli-responsive ONs reported in this review, no application for controlling gene expression has been shown, but a certain potential in this field could be demonstrated. Additionally, other applications in different domains have been mentioned to extend the interest in such molecules

α,β-D-CNA preorganization of unpaired loop moiety stabilizes DNA hairpin

International audienceDNA hairpin structures can be stabilized by introduction of constraint on the sugar phosphate backbone within the unpaired loop moiety by means of dinucleotides locked with an alpha torsion angle in gauche(+) configuration

A versatile post-synthetic method on a solid support for the synthesis of RNA containing reduction-responsive modifications

International audienceAn original post-synthetic method on a solid support was developed to introduce various disulfide bond containing groups at the 2'-OH of oligoribonucleotides (RNAs). It is based on a thiol disulfide exchange reaction between several readily accessible alkyldisulfanyl-pyridine derivatives and 2'-O-acetylthiomethyl RNA in the presence of butylamine. By this strategy, diverse 2'-O-alkyldithiomethyl RNAs were obtained. These modifications provided high nuclease resistance to RNA and were easily removed with glutathione treatment, thus featuring a potential use for siRNA prodrugs

Chemical synthesis of RNA with base-labile 2'-O-(pivaloyloxymethyl)-protected ribonucleoside phosphoramidites.

International audienc

Conjugation of Small Molecules to RNA Using a Reducible Disulfide Linker Attached at the 2′-OH Position through a Carbamate Function

International audienc

Probing the Active Site of the Deoxynucleotide N-Hydrolase Rcl Encoded by the Rat Gene c6orf108.

International audienceRcl is a potential anti-angiogenic therapeutic target that hydrolyzes the N-glycosidic bond of 2'-deoxyribonucleoside 5'-monophosphate, yielding 2-deoxyribose 5-phosphate and the corresponding base. Its recently elucidated solution structure provided the first insight into the molecular basis for the substrate recognition. To facilitate the development of potent and specific inhibitors of Rcl, the active site was probed by site-directed mutagenesis and by the use of substrate analogs. The nucleobase shows weak interactions with the protein, and the deoxyribose binding pocket includes the catalytic triad Tyr-13, Asp-69, and Glu-93 and the phosphate binding site Ser-87 and Ser-117. The phosphomimetic mutation of Ser-17 to Glu prevents substrate binding and, thus, abolishes the activity of Rcl. The synthetic ligand-based analysis of the Rcl binding site shows that substitutions at positions 2 and 6 of the nucleobase as well as large heterocycles are well tolerated. The phosphate group at position 5 of the (deoxy)ribose moiety is the critical binding determinant. This study provides the roadmap for the design of small molecules inhibitors with pharmacological properties

Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers

International audienceCo-delivery systems of siRNA and chemotherapeutic drugs have been developed as an attractive strategy to optimize the efficacy of chemotherapy towards cancer cells with multidrug resistance. In these typical systems, siRNAs are usually associated to drugs within a carrier but without covalent interactions with the risk of a premature release and degradation of the drugs inside the cells. To address this issue, we propose a covalent approach to co-deliver a siRNA-drug conjugate with a redox-responsive self-immolative linker prone to intracellular glutathione-mediated disulfide cleavage. Herein, we report the use of two disulfide bonds connected by a pentane spacer or a p-xylene spacer as self-immolative linker between the primary amine of the anticancer drug doxorubicin (Dox) and the 2 ' -position of one or two ribonucleotides in RNA. Five Dox-RNA conjugates were successfully synthesized using two successive thiol-disulfide exchange reactions. The Dox-RNA conjugates were annealed with their complementary strands and the duplexes were shown to form an A-helix sufficiently stable under physiological conditions. The enzymatic stability of Dox-siRNAs in human serum was enhanced compared to the unmodified siRNA, especially when two Dox are attached to siRNA. The release of native Dox and RNA from the bioconjugate was demonstrated under reducing conditions suggesting efficient linker disintegration. These results demonstrate the feasibility of making siRNA-drug conjugates via disulfide-based self-immolative linkers for potential therapeutic applications

Stabilization of hairpins and bulged secondary structures of nucleic acids by single incorporation of α,β-D-CNA featuring a gauche(+) alpha torsional angle

A constrained dinucleotide unit featuring a gauche(+) alpha torsional angle configuration was used to stabilize DNA hairpin or bulged structures. Large five nucleotides having looped hairpin structures can be stabilized up to +5 degrees C. Depending on the nature of the closing base pair, the increased hairpin stability can be reached through loop moiety preorganization or stem rearrangement induced on the first two base pairs. With an alpha gauche(+) constraint either within or opposite to the bulge, the larger bulged structures had better stabilization

α,β-D-CNA induced rigidity within oligonucleotides

International audienceIntroduction of α,β-D-CNA featuring canonical values of the torsional angles α and β within oligonucleotides leads to an overall stabilization and improved rigidity of the duplex DNA as demonstrated by UV experiments, circular dichroism and corroborated by molecular dynamics simulations