Bioconjugation of Small Molecules to RNA Impedes Its Recognition by Toll-Like Receptor 7

Abstract

A fundamental mechanism of the innate immune system is the recognition, via extra- and intracellular pattern-recognition receptors, of pathogen-associated molecular patterns. A prominent example is represented by foreign nucleic acids, triggering the activation of several signaling pathways. Among these, the endosomal toll-like receptor 7 (TLR7) is known to be activated by single-stranded RNA (ssRNA), which can be specifically influenced through elements of sequence structure and posttranscriptional modifications. Furthermore, small molecules TLR7 agonists (smTLRa) are applied as boosting adjuvants in vaccination processes. In this context, covalent conjugations between adjuvant and vaccines have been reported to exhibit synergistic effects. Here, we describe a concept to chemically combine three therapeutic functions in one RNA bioconjugate. This consists in the simultaneous TLR7 stimulation by ssRNA and smTLRa as well as the therapeutic function of the RNA itself, e.g., as a vaccinating or knockdown agent. We have hence synthesized bioconjugates of mRNA and siRNA containing covalently attached smTLRa and tested their function in TLR7 stimulation. Strikingly, the bioconjugates displayed decreased rather than synergistically increased stimulation. The decrease was distinct from the antagonistic action of an siRNA bearing a Gm motive, as observed by direct comparison of the effects in the presence of otherwise stimulatory RNA. In summary, these investigations showed that TRL7 activation can be impeded by bioconjugation of small molecules to RNA.

Multivalency Beats Complexity: A Study on the Cell Uptake of Carbohydrate Functionalized Nanocarriers to Dendritic Cells

“…A key study by Holla and Skerra revealed that simple mannose-units, and especially branched oligomannosidic structures, in particular a trimannose structure (3,6-di-(α-d-mannopyranosyl)-α-d-mannopyranose) as well as fucose-containing saccharides, in particular a disaccharide (3-(β-l-fucopyranosyl)-2-acetamido-2-deoxy-β-d-glucopyranose), are recognized by DC-SIGN [25]. Based on these observations, we selected four different potential ligands for DC-SIGN that carry a poly(ethylene glycol) (PEG)-based, azide-terminated linker to enable covalent binding through azide-alkyne click reactions to nanocarriers as the target structures for total synthesis (Figure 1) A simple d-mannose monosaccharide 1 and the mentioned trimannose structure 2, which were available from earlier studies [4,26,27], as well as a fucose containing disaccharide 3 and a mannose-terminated glycodendron 4, were chosen as potential ligands. The synthesis of the fucose disaccharide 3 started from N-acetylglucosamine (5), which was deprotonated at the most acidic 1-OH position and then was β-selectively alkylated with progargyl bromide in 52% yield (Scheme 1, top) [29].…”

“…The ester moieties were cleaved using TFA and then functionalized with amine-azide linker 13 to give the dendron core 14 under standard amide coupling conditions in quantitative yield [32]. Mannose alkyne 15, which was synthesized according to a previous report [26], was coupled to the dendron core 14 using the previously mentioned CuAAC conditions in 75% yield. The tertiary amine in the dendron core was then deprotected using a photochemical cleavage of the o-nitrobenzyl moiety upon irradiation with UV-A light [33].…”

Double methylation of tRNA-U54 to 2′-O-methylthymidine (Tm) synergistically decreases immune response by Toll-like receptor 7

“…Further studies may identify additional contributions of single modifications, but we would like to point out some of our results with synthetic modifications in this context. We recently reported a generic ‘shielding’ effect of a number of synthetic RNA modifications with no discernible common denominator in terms of chemical structure ( 41 ). The conclusion from those studies might also apply to heavily modified eukaryotic tRNA.…”

A Photoredox-Catalyzed Four Component Reaction for the Atom-Efficient Synthesis of Complex Secondary Amines

“…[27] Under the mild reaction conditions, typical protecting groups such as Fmoc (15,41), acetate (43, 44) and benzoate esters (45, 46) and especially acetals (47, 48) and thioketals (31) remain untouched. The carbonyl group in 2-acetylpyridine, which could potentially form a ketimine, is well tolerated (28). Besides functionalized benzaldehyde derivatives, electron-deficient pyridine-2-carbaldehyde (38) or electron-rich thiophene-2carbaldehyde (39, 32) react smoothly in this sequenced radical cascade.…”

“…The 4-CR of the ibuprofen ester of p-hydroxybenzaldehyde furnished compound 40 in 46% yield. Even complex carbohydrates such as di-and trisaccharide derivatives (45−48), suitable for targeting dendritic cells, [28] reacted smoothly and provided synthetically useful yields of the desired coupling products.…”