Fluorescent Properties of Carboxyfluorescein Bifluorophores.

Bright fluorescent probes with enhanced intensities in the fluorescein channel are of great value for plenty of biological applications. To design effective probes one should introduce as many as possible fluorophores to the biomolecule while leaving its native structure as intact as possible. To reach this compromise, we designed and synthesized fluorescein bifluorophores on the 3,5-diaminobenzoic acid scaffold, which allows for insertion of two fluorophores at one modification site of a biomolecule. Rigid structure of the branching linker group allows to minimize self-quenching the fluorophores. However, despite the structure similarities of fluorescein isomers (5-FAM and 6-FAM), different photophysical behavior was observed for the corresponding bifluorophores. Here we made efforts to get insight into these effects with the focus on the media viscosity impact.

Novel Cluster and Monomer-Based GalNAc Structures Induce Effective Uptake of siRNAs in Vitro and in Vivo.

GalNAc conjugation is emerging as a dominant strategy for delivery of therapeutic oligonucleotides to hepatocytes. The structure and valency of the GalNAc ligand contributes to the potency of the conjugates. Here we present a panel of multivalent GalNAc variants using two different synthetic strategies. Specifically, we present a novel conjugate based on a support-bound trivalent GalNAc cluster, and four others using a GalNAc phosphoramidite monomer that was readily assembled into tri- or tetravalent designs during solid phase oligonucleotide synthesis. We compared these compounds to a clinically used trivalent GalNAc cluster both in vitro and in vivo. In vitro, cluster-based and phosphoramidite-based scaffolds show a similar rate of internalization in primary hepatocytes, with membrane binding observed as early as 5 min. All tested compounds provided potent, dose-dependent silencing, with 2-4% of injected dose recoverable from liver after 1 week. The two preassembled trivalent GalNAc clusters showed higher tissue accumulation and gene silencing relative to di-, tri-, or tetravalent GalNAc conjugates assembled via phosphoramidite chemistry.

Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N-Acetylgalactosamine Clusters.

We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt)3 as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.

Synthesis of GalNAc-Oligonucleotide Conjugates Using GalNAc Phosphoramidite and Triple-GalNAc CPG Solid Support.

GalNAc oligonucleotide conjugates demonstrate improved potency in vivo due to selective and efficient delivery to hepatocytes in the liver via receptor-mediated endocytosis. GalNAc-siRNA and GalNAc-antisense oligonucleotides are at various stages of clinical trials, while the first two drugs were already approved by FDA. Also, GalNAc conjugates are excellent tools for functional genomics and target validation in vivo. The number of GalNAc residues in a conjugate is crucial for delivery as cooperative interaction of several GalNAc residues with asialoglycoprotein receptor enhances delivery in vitro and in vivo. Here we provide a robust protocol for the synthesis of triple GalNAc CPG solid support and GalNAc phosphoramidite, synthesis and purification of RNA conjugates with multiple GalNAc residues either to 5'-end or 3'-end and siRNA duplex formation.