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. Notably, DOPE associated with EVs quickly, and the products remained stable under several challenging conditions. Moreover, conjugating fluorophores, receptor-targeting peptides or albumin-binding molecules with DOPE enabled tracking the cellular uptake, enhanceing the cellular uptake or extending the circulation time in mice of engineered EVs , respectively. Taken together, this study reports an efficient lipid anchor for exogenous engineering of EVs and further showcases its versatility for the functionalization of EVs.

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

2'-O-(N-(Aminoethyl)carbamoyl)methyl (2'-O-AECM)-modified oligonucleotides (ONs) and their mixmers with 2'-O-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. It is therefore particularly interesting and unprecedented that the 2'-O-AECM ONs are shown to have efficient splice-switching activity even without inclusion of PS linkages and found to be as effective as 2'-OMe PS ONs. Importantly, the PS linkages can be partially included, without any significant reduction in splice-switching efficacy. This suggests that AECM modification has the potential to be used in balancing the PS content of ONs. Furthermore, conjugation of 2'-O-AECM ONs to an endosomal escape peptide significantly increased splice-switching suggesting that this effect could possibly be due to an increase in uptake of ON to the site of action.

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. This report describes the simplified methodology allowing for multiple labeling of ONs on a solid support and is compatible with phosphodiester as well as phosphorothioate (PS) ONs. The current approach utilizes two novel alkyne- and amino-functionalized linker phosphoramidites that can be readily synthesized from a common aminodiol intermediate in three steps. The combination of new linkers provides orthogonal functionalities, which allow for multiple attachments of similar or varied moieties. The linkers are incorporated into ONs during automated solid-phase ON synthesis, and the conjugation with functional entities is achieved by either amide bond formation or by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The versatility of the approach is demonstrated by the synthesis of 5'-site ON multiconjugates with small molecules, peptides, and fatty acids as well as in the preparation of an internal peptide-ON conjugate.

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 × Me2 S) as a mediator for the click reaction. The solid-supported ONs can be readily transformed into "clickable ONs" by on-line addition of an alkyne-containing linker that subsequently can react with an azido-containing moiety (e.g., a peptide) in the presence of CuBr × Me2 S. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Conjugation on solid support Support Protocol: Removal of 4,4'-dimethoxytrityl group from amino linker Basic Protocol 2: Removal of protecting groups and cleavage from solid support Basic Protocol 3: HPLC purification.

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. Conjugation reaction is efficient in millimolar concentration and submicromolar amounts at ambient temperature. The route for POC preparation involves two subsequent conjugation steps: to solid-supported ONs containing a 5'-amino modifier (1) the triple bond donor (p-(N-propynoylamino)toluic acid (PATA), p-([2-(propynyloxy)acetamido]methyl)benzoic acid (PAMBA) or 2-(propynyloxy)acetic acid (PAA)) is first coupled and then (2) an azido-functionalized peptide is attached via a triazole linkage by copper(I) catalyzed Huisgen 1,3-dipolar cycloaddition. The fragment-conjugated POC is released from the solid support by concentrated ammonia. The method gives high conversion of ON to the POC and only involves a single purification step after complete assembly and release from the solid support. The synthesis is flexible and designed to utilize commercially available oligonucleotide and peptide derivatives without the need for specific automated synthesizers.

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. However, the majority of potential oligonucleotide (ON) therapeutics in clinical trials are carrying phosphorothioate (PS) linkages, and this robust conjugation method is not yet established for these ONs due to a general concern of Cu-S interaction. Here, we developed a method allowing for efficient conjugation of peptides to PS oligonucleotides. The method utilizes solid supported oligonucleotides that can be readily transformed into "clickable ONs" by simple linker conjugation and further reacted with an azido containing moiety (e.g., a peptide) using the CuBr × Me2S complex as a superior catalyst in that reaction. This study opens the way for further development of PS oligonucleotide-conjugates by means of efficient 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. Sequential solid-phase synthesis of oligonucleotide conjugates containing three attached entities was performed using an acetylated form of MIF peptide conjugated to azido linker, achieving high conversions at each unit addition. In addition, to show the versatility of the method, oligonucleotide conjugates with several different classes of compounds were synthesized. Each conjugate containing three different entities, whose structure and function varied (e.g., sugars, peptides, fluorescent labels, and m3G-Caps).

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. In addition to achieving sequence specific dsDNA binding, we have investigated the potential of these compounds to enter cells. Confocal microscopy and flow cytometry show the beneficial influence of the steroid on cell uptake. This unique synthetic model of the bZIP TF thus combines sequence specific dsDNA binding properties with enhanced cell-uptake. Given the unique properties of deoxycholic acid and the convergent nature of the synthesis, we believe this work represents a key achievement in the field of TF mimicry.

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. Here we report on synthesis of three m3G-CAP derivatives with a 'native' (m3GpppAOMe) as well as with a methylenephosphonate stabilized triphosphate bridge (m3GpCH2ppAOMe, m3GppCH2pAOMe) and the investigation of the enzymatic stability of these compounds in 10% (v/v) fetal bovine serum (FBS) and cytosolic extract from HeLa cells, thus mimicking in vivo conditions. Our results indicate that introduction of methylene group between the ß and γ phosphates in m3GpCH2ppAOMe improves to some extent stability of this analogue in 10% serum but does not prolong life of this compound in the cytosolic extract. In contrast the stabilization introduced between α and ß phosphates in m3GppCH2pAOMe offers threefold longer life in 10% serum and almost complete protection in cytosolic extract.

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. Its exclusive affinity to fluorescent streptavidin/avidin proteins allows ready and specific detection. As a consequence methods for the attachment of biotin to various biological targets are of high importance, especially when they are very selective and can also proceed in water. One useful method is Hüisgen dipolar [3+2]-cycloaddition, commonly referred to as “click chemistry”. As we reported recently, the activated triple bond donor p-(N-propynoylamino)toluic acid (PATA) gives excellent results when used for conjugations at submicromolar concentrations. Thus, we have designed and synthesized two biotin linkers, with different lengths equipped with this activated triple bond donor and we proceeded with biotinylation of oligonucleotides and C-myc peptide both in solution and on solid support with excellent yields of conversion.