Surface display of functional moieties on extracellular vesicles using lipid anchors.

Extracellular vesicles (EVs) are efficient natural vehicles for intercellular communication and are under extensive investigation for the delivery of diverse therapeutics including small molecule drugs, nucleic acids, and proteins. To understand the mechanisms behind the biological activities of EVs and develop EV therapeutics, it's fundamental to track EVs and engineer EVs in a customized manner. In this study, we identified, using single-vesicle flow cytometry and microscopy, the lipid DOPE (dioleoyl phosphatidyl ethanolamine) as an efficient anchor for isolated EVs.

2'--(-(Aminoethyl)carbamoyl)methyl Modification Allows for Lower Phosphorothioate Content in Splice-Switching Oligonucleotides with Retained Activity.

2'--(-(Aminoethyl)carbamoyl)methyl (2'--AECM)-modified oligonucleotides (ONs) and their mixmers with 2'--methyl oligonucleotides (2'-OMe ONs) with phosphodiester linkers as well as with partial and full phosphorothioate (PS) inclusion were synthesized and functionally evaluated as splice-switching oligonucleotides in several different reporter cell lines originating from different tissues. This was enabled by first preparing the AECM-modified A, C, G and U, which required a different strategy for each building block. The AECM modification has previously been shown to provide high resistance to enzymatic degradation, even without PS linkages.

New Alkyne and Amine Linkers for Versatile Multiple Conjugation of Oligonucleotides.

Oligonucleotide (ON) conjugates are increasingly important tools for various molecular diagnostics, nanotechnological applications, and for the development of nucleic acid-based therapies. Multiple labeling of ONs can further equip ON-conjugates and provide improved or additional tailored properties. Typically, the preparation of ON multiconjugates involves additional synthetic steps and/or manipulations in post-ON assembly.

Copper-Catalyzed Huisgen 1,3-Dipolar Cycloaddition Tailored for Phosphorothioate Oligonucleotides.

An efficient method for attachment of a variety of reporter groups to oligonucleotides (ONs) is copper (I) [Cu(I)]-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition ("click reaction"). However, in the case of ONs with phosphorothioate modifications as internucleosidic linkages (PS-ONs), this conjugation method has to be adjusted to be compatible with the sulfur-containing groups. The method described here is adapted for PS-ONs, utilizes solid-supported ONs, and implements the Cu(I) bromide dimethyl sulfide complex (CuBr × Me S) as a mediator for the click reaction.

Attachment of Peptides to Oligonucleotides on Solid Support Using Copper(I)-Catalyzed Huisgen 1,3-Dipolar Cycloaddition.

In vivo bioavailability and delivery of nucleic acids to the site of action is a severe limitation in oligonucleotide (ON) therapeutics. Equipping the ONs with cell penetrating, homing or endosomal escape peptides can enhance specificity and/or uptake efficiencies. We describe here a general procedure for the preparation of peptide-oligonucleotide conjugates (POCs) on solid support utilizing a novel activated alkyne containing linker which enhances the Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition.

Efficient Conjugation to Phosphorothioate Oligonucleotides by Cu-Catalyzed Huisgen 1,3-Dipolar Cycloaddition.

Improving oligonucleotide delivery is critical for the further development of oligonucleotide-based therapeutics. Covalent attachment of reporter molecules is one of the most promising approaches toward efficient oligonucleotide-based therapies. An efficient methods for the attachment of a variety of reporter groups is Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition.

Enabling Multiple Conjugation to Oligonucleotides Using "Click Cycles".

An efficient method for the synthesis of multiply functionalized oligonucleotides (ONs) utilizing a novel H-phosphonate alkyne-based linker for multiple functionalization (LMF) is developed. The strategy allows for the conjugation of various active entities to oligonucleotide through the postsynthetic attachment of LMF at the 5'-terminus of ONs using H-phosphonate chemistry followed by conjugation of various entities via [3 + 2] copper(I) catalyzed cycloaddition in a stepwise manner. Each cycle is composed of attachment of the LMF followed by a click reaction with azido-containing units.

Sequence-selective DNA recognition and enhanced cellular up-take by peptide-steroid conjugates.

Several GCN4 bZIP TF models have previously been designed and synthesized. However, the synthetic routes towards these constructs are typically tedious and difficult. We here describe the substitution of the Leucine zipper domain of the protein by a deoxycholic acid derivative appending the two GCN4 binding region peptides through an optimized double azide-alkyne cycloaddition click reaction.

Synthesis and evaluation of stability of m3G-CAP analogues in serum-supplemented medium and cytosolic extract.

Increased efficiency in splice-correction (splice-switching) has been shown by use of a synthetic RNA 5'-end nuclear localization signal composed of an m3G-CAP. Use of the m3G-CAP as an NLS signal for therapeutic compounds in vivo is likely to require additional stability towards enzymatic degradation. For this reason introduction of stabilizing modifications into the triphosphate bridge may be beneficial.

Synthesis of biotin linkers with the activated triple bond donor [p-(N-propynoylamino)toluic acid] (PATA) for efficient biotinylation of peptides and oligonucleotides.

Biotin is an important molecule for modern biological studies including, e.g., cellular transport.