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An LNA-amide modification that enhances the cell uptake and exercise of phosphorothioate exon-skipping oligonucleotides


LNA-amide monomer synthesis is environment friendly and scalable

Environment friendly synthesis of chemically modified oligonucleotides is important to gas basic research and therapeutic functions. We have now devised a technique which allows the easy meeting of oligonucleotides containing LNA-flanked amides which might be simply automated and scaled up. A most of eight monomers are required to make any sequence that incorporates non-contiguous amide linkages (4 carboxylic acids and 4 amines).

Attaching the required ethanoic acid moiety to the three′-carbon of the LNA sugar is difficult because it requires the elimination of the three′-oxygen and the formation of a C-C bond. Nevertheless, we have been in a position to produce the required 5′-dimethoxytrityl (DMT)-protected 3′-ethanoic acid LNA-monomers in 8 steps from 1, the identical quantity as for standard LNA phosphoramidites40 (Fig. 2a). Furthermore, we did this with minimal chromatography. We first constructed the sugar with the C3′-ester 5, earlier than the addition of the nucleobase. This strategy avoids problems related to forming a C-C bond on the 3′-side of a nucleoside utilizing the Barton-McCombie response23,24,25,41 or the hydrogenation step if a Wittig response is used42,43, which might in the end restrict the vary of heterocyclic bases that may be added. Key intermediate 5 was readily ready on a a number of gram scale in 81% total yield from commercially out there 1 with out the necessity for chromatographic separation as follows. Hydrogenolysis of compound 1 afforded alcohol 2 which was subsequently oxidised to present compound 3. Olefination of 3 with (carbethoxymethylene)triphenylphosphorane (Wittig response) selectively yielded 4 because the (E)-stereoisomer (Fig. 2b). Catalytic hydrogenation of 4 utilizing Pd/C and H2 gave 5 as a single stereoisomer (Fig. 2b). This stereoselectivity was predicted as a result of the 1,2-O-isopropylidene teams on the α-face of furanosyl carbohydrate derivatives direct incoming H2 to the β-face44. Key intermediate 5 was then transformed to the 1,2-di-O-acetate glycosyl donor 6 following a process reported by Arzel et al.45, avoiding the formation of a lactone which occurred when the acetonide was cleaved within the presence of water.

Fig. 2: LNA-ethanoic acids and different monomers used on this examine.
figure 2

a Synthesis of the DMT-protected LNA-ethanoic acids 9ae. b X-ray crystal buildings confirming the (E)-configuration in 4 and the stereochemistry on the 3′-carbon in 5 (particulars in ‘Small molecule crystallography’ part within the Supplementary Data). c Phosphoramidites 1047 and 11 (commercially out there) and DNA-ethanoic acid monomer 1248,49 are used to synthesise oligonucleotides. BSA bis(trimethylsilyl)acetamide, TMSOTf trimethylsilyl trifluoromethanesulfonate, DMAP 4-dimethylaminopyridine, DMT 4,4′-dimethoxytrityl, MMT 4-monomethoxytrityl.

The pathways to every monomer then diverged, with Vorbrüggen circumstances40,46 utilised for the addition of the nucleobases to entry 7ae. Subsequent simultaneous unmasking of the three′-carboxyl and a pair of′-hydroxyl teams by remedy with hydroxide, adopted by cyclisation to kind the two′-4′-oxymethylene bridge, then 5′-mesyl deprotection, gave the hydroxy-LNA-acid compounds 8ae. The progress of the response was fast; mesyl deprotection utilizing hydroxide ion conventionally requires a number of days below reflux circumstances. We postulate that the acceleration in charge is because of neighbouring group participation whereby the carboxylate anion displaces the 5′-mesyl group, forming a lactone that’s subsequently opened by hydrolysis (Supplementary Fig. 1). Lastly, we handled the ensuing hydroxy-acids 8ae with 4,4′-dimethoxytrityl chloride (DMT-Cl) in pyridine to present the DMT-protected LNA analogue nucleosides 9ae. Utilizing this technique we have been in a position to entry all 4 canonical nucleoside analogues together with the 5-methylcytidine monomer which is used instead of cytidine in antisense experiments to extend goal affinity and enhance different therapeutic properties18. Moreover, we required 5′-MMT-amino LNA phosphoramidite 10. While this had been beforehand synthesised47 we selected to develop a extra environment friendly route (Supplementary Fig. 2). Commercially out there 5′-MMT-amino dT 11 and 5′-DMT thymidine-3′-ethanoic acid 1248,49 (Fig. 2c) have been additionally required to allow us to make oligonucleotides to match the properties of DNA-amide with these of LNA-amide.

Chimeric oligonucleotides may be synthesised in excessive purity

Our oligonucleotide synthesis technique is proven in Fig. 3. A phosphoramidite monomer with an MMT-protected 5′-amino group, both LNA 1040 or deoxythymidyl 11, is added to the oligonucleotide, and the amine is deprotected utilizing trichloroethanoic acid. An LNA-acid (or DNA-acid50) monomer is coupled to the free amine utilizing PyBOP activating agent within the presence of a non-nucleophilic base (N-methylmorpholine) to kind the amide bond. Oligonucleotide synthesis is then resumed, beginning with the elimination of the DMT group. The method is repeated to put in a number of non-contiguous amides in the identical oligonucleotide. To reveal this, DMT-protected LNA acids 9ae, phosphoramidites 10 and 11, and DNA-acid 1248,49 (Fig. 2), have been used to synthesise a number of oligonucleotides, a few of which comprise a number of additions of LNA-amide, 2′-OMe sugars and PS linkages (Supplementary Desk 1). In all instances, we obtained the oligonucleotides in excessive purity. Excessive-performance liquid chromatography (HPLC) and mass spectrometry demonstrated the excessive incorporation effectivity for the DMT-protected LNA acids 9ae (Supplementary Figs. 37).

Fig. 3: Synthesis of (LNA)-amide-phosphodiester chimeric oligonucleotides.
figure 3

a Environment friendly solid-phase synthesis of amide-phosphodiester chimaeras. Dashed traces point out the presence or absence of two′-4′-methylene bridge. PyBOP benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate. b Reversed-phase HPLC chromatogram. X-axis = time in minutes. y-axis = absorbance at 260 nm. c Electrospray ES- mass spectrum of a crude LNA-amide chimeric oligonucleotide (ON14). X-axis = mass Y-axis = relative depth. Required mass = 5438.7 Da, Discovered mass = 5439.0 Da.

LNA sugars stabilise duplexes containing the amide spine

To guage the flexibility of the LNA-amide mixture to bind to complementary RNA with excessive affinity we synthesised a collection of 13-mer ONs with a central amide with a T-T sequence. These constructs have been composed of both no LNA (ON1DNA-Am-DNA), an LNA 5′ to the amide (ON2LNA-Am-DNA), an LNA 3′ to the amide (ON3DNA-Am-LNA), or LNA on either side of the amide (ON4LNA-Am-LNA, Fig. 4a). Controls with out amide (ON5LNA-LNA and ON6DNAcontrol) have been additionally made. We in contrast duplex denaturation temperatures (Tms) after hybridisation with DNA and RNA complementary strands (Fig. 4b, Supplementary Desk 2 and Supplementary Figs. 8, 9). ON2LNA-Am-DNA produced a major improve in DNA:RNA hybrid stability, (+3.0 ˚C) in comparison with the unmodified ON6DNAcontrol, and a rise of +3.4 C in comparison with ‘amide solely’ ON1DNA-Am-DNA. Importantly, ON4LNA-Am-LNA, through which the amide is surrounded by LNA sugars, gave the best improve in stability (+5.1 ˚C). It’s noteworthy that ON2LNA-Am-DNA and ON4LNA-Am-LNA present the primary examples of duplex stabilisation by an LNA sugar hooked up to a non-phosphorus synthetic DNA spine. The RNA goal selectivity of LNA-amide-containing ONs was glorious; a single mismatched base pair drastically diminished duplex stability, in some instances by >14 ˚C (Supplementary Desk 2 and Supplementary Figs. 1013) In abstract, an amide linkage flanked by LNA on either side provides robust DNA:RNA duplex stabilisation and good mismatch discrimination.

Fig. 4: Combining LNA and AM1 improve affinity for complementary RNA.
figure 4

a The buildings of every of the LNA AM1 mixtures studied. Sequence d-CGACGCTTGCAGC (the place the underlined area signifies the modification place) b UV-melting curve derivatives of the ONs from panel a in duplexes with complementary RNA. c UV-melting curve derivatives reveal that the stabilisation conferred by the LNA-amide linkage in opposition to complementary RNA is basic and cumulative. Circumstances: 10 mM phosphate buffer, 200 mM NaCl, pH 7.0. Sequences in Supplementary Desk 1.

In duplexes with DNA targets, ONs with all mixtures of LNA and DNA sugars across the amide linkage have been barely destabilising (between −0.1 to −2.6 ˚C), indicating the selectivity of the amide linkage for complementary RNA. The stabilisation induced by the LNA-amide mixture is cumulative and basic (Fig. 4c, Supplementary Desk 3 and Supplementary Figs. 1417). In a biologically related sequence context, 4 LNA-amides improve duplex stability in opposition to complementary RNA by a formidable 13.0 ˚C in comparison with solely 5.1 ˚C for the DNA goal. This massive distinction is necessary when growing oligonucleotides to work together with RNA. Oligonucleotides for in vivo research often comprise 2′-OMe modified sugars and/or phosphorothioate backbones to forestall degradation by nucleases. In such oligonucleotides, the mixture of LNA and amide additionally drastically improve duplex stability (Supplementary Desk 3).

Combining LNA and amide gives robust nuclease resistance

Therapeutic oligonucleotides should stay steady in cells for extended intervals to stay lively. To guage whether or not the mixture of LNA and amide confers higher nuclease resistance than LNA alone, we incubated unmodified DNA (ON15DNA/17PO) and DNA with 4 LNA-amide linkages (ON14 DNA/4LAL/13PO) in a 1:1 combination of phosphate-buffered saline (PBS) and foetal bovine serum (FBS) to imitate the in vivo atmosphere and in contrast it to the equal assemble with LNA however with out amide linkages (ON25DNA/8LNA/17PO, Supplementary Fig. 18). The outcomes present that the mixture of LNA and amide confers excessive resistance to nucleases. Each the oligonucleotides missing amide linkages had partially degraded inside 1 h whereas ON14DNA/4LAL/13PO remained intact after 8 h. Apparently ON14 exhibits stability at its 3′-end, although this area has an unmodified sugar-phosphate trimer. The three′-terminal pentamer area, nevertheless, is very modified. It has a diminished cost as a result of amide linkage and in addition incorporates two LNA sugars. It might due to this fact not be recognised by nucleases. This enhanced stability additional illustrates some great benefits of eradicating cost and together with modified sugars in antisense oligonucleotides.

X-ray buildings of the LNA-amide modification in DNA-RNA hybrids

We solved a number of X-ray buildings to find out the results of LNA and amide modifications on duplex construction and conformation. These are the primary crystal buildings of DNA:RNA hybrids that comprise amide linkages. An amide-modified RNA:RNA duplex was analysed beforehand, however this had amides in each strands surrounded by a number of mismatched base pairs which can’t exist outdoors the stable state at ambient temperature27. The sequence of the modified DNA:RNA hybrid duplexes (Supplementary Desk 4) was primarily based on the corresponding unmodified model (d-CTTTTCTTTG/rCAAAGAAAAG)51 (the placement of the amide is underlined). Good high quality crystals diffracting between 2.5–2.8 Å decision have been obtained for the DNA:RNA hybrid through which the DNA strand incorporates an amide linkage flanked by DNA on either side, LNA on either side and with LNA solely on the 5′-side (Supplementary Fig. 19). The unmodified DNA:RNA duplex was additionally studied. The info assortment and refinement statistics are given in Supplementary Desk 5. Electron density maps on the modification place for the 4 nucleic acid crystal buildings reported on this manuscript are given in Supplementary Fig. 20. The hybrids with amide and LNA-amide backbones (Fig. 5a) are structurally similar to the unmodified duplex (all-atom RMSD 0.4 Å) as proven by their superimposition (Fig. 5a and Supplementary Fig. 21). All buildings undertake the A-conformation with sugar puckers clustering round C3′-endo (Supplementary Fig. 22). As anticipated, all duplexes are stabilised by canonical Watson–Crick base pairs, indicating that the thermodynamic enhancements as a result of LNA-amide backbones will not be resulting from uncommon adjustments in hydrogen bonding interactions. In settlement with the DNA:RNA hybrid NMR construction by Rosners22 through which the DNA strand contained a number of amides, our X-ray research point out that the amide linkage is a detailed mimic of the phosphodiester spine (Fig. 5b). Each are four-atom linkages, therefore related in size, and the amide carbonyl is oriented in the identical route as one of many phosphodiester P-O bonds.

Fig. 5: X-ray buildings of amide and LNA-amide-modified DNA:RNA duplexes.
figure 5

a Structural identification of amide and LNA-amide modifications and the torsion angles of the amide spine (5′ε → 3′δ). Pink steps present the modification place (left) and the overlay of all buildings exhibits clear similarities (proper). b Spine overlay evaluating the LNA-amide-LNA step in ON30xLNA-Am-LNA (purple) with the phosphodiester in ON26xDNA (gray). c Overlay of all amide backbones with or with out LNA modifications (gentle blue = ON28xDNA-Am-DNA, darkish blue = ON29xLNA-Am-DNA, purple = ON30xLNA-Am-LNA).

In Fig. 5c, the buildings of all amide backbones are overlaid to evaluate the results of the LNA modifications. Between every construction, the orientation of the spine is constant, directing the amide oxygen into the most important groove. Different atomic positions of the backbones additionally present shut similarity, and the presence of three′-LNA causes no important distortion. 5′-LNA does nevertheless trigger some structural displacement; the 5′-sugars within the LNA-amide-DNA and LNA-amide-LNA buildings are shifted barely outwards in comparison with the DNA-amide-DNA and unmodified strands. Regardless of this, the positioning of the amide spine stays constant between every construction. The amide adopts the anticipated trans-conformation, and LNA on the 5′-side of the amide has little impact on spine torsion angles (Fig. 5c and Supplementary Fig. 23). In abstract, mixed LNA and amide modifications have minimal impact on the duplex construction, and are glorious mimics of pure phosphodiesters.

Combining LNA-amide and PS enhances gymnotic supply

In a preliminary examine, we’ve got evaluated the organic exercise of the LNA-amide mixture utilizing the HeLa pLuc/705 cell line52 that carries a luciferase-encoding gene interrupted by a mutated ß-globin intron52. This mutation creates a 5′-splice website which prompts a cryptic 3′-splice website, leading to incorrect mRNA splicing and the manufacturing of non-functional luciferase. An oligonucleotide that hybridises to the mutant 5′-splice website prevents incorporation of the aberrant intron, restoring the pre-mRNA splicing to supply purposeful luciferase, which is quantified by luminometry. Oligonucleotides complementary to this aberrant splice website have been synthesised with mixtures of various modifications to find out their particular person results (Supplementary Desk 3). ON14DNA/4LAL/13PO, ON162′OMe/4LAL/13PO, and ON182′OMe/4LAL/13PS have been designed to guage LNA-amide with the DNA, 2′-OMe/phosphodiester, and a pair of′-OMe/phosphorothioate backbones respectively, and ONs 14 and 16 have been included to find out the diploma to which LNA-amide influences supply, exercise and toxicity within the absence of PS linkages. LNA and amide linkages are incompatible with RNase-H so there isn’t a threat of ON14 or ON16 inadvertently destroying the RNA goal2. Three controls have been included: ON202′OMe/17PS (which represents the gold commonplace within the assay) to find out whether or not LNA-amide enhances organic exercise52, ON172′OMe/17PO to guage the results of the PS linkage independently of LNA or amide linkages, and ON192′OMe/8LNA/17PS with LNA sugars however no amide linkages to find out the results of the improved duplex stability brought on by LNA. A scrambled management with a 2′-OMe/PS spine was additionally included to find out off-target results (ON312′OMe/17PS scrambled, Fig. 6).

Fig. 6: LNA-flanked amides and PS linkages improve gymnotic supply, exon-skipping exercise and cut back toxicity.
figure 6

In all instances, luciferase exercise was measured and normalised to each protein amount and untreated cells. Experimental particulars and sequences are given within the Supplementary Data. a HeLa pLuc/705 cells have been transfected at 100 nM ON utilizing LF2000. b Dose-response for lively ONs in a. c Cells have been handled with ONs and not using a transfection agent. d Viability of the HeLa pLuc/705 cells following lipofection with ONs utilizing LF2000 decided utilizing WST-1 assay. For all graphs (ad) knowledge have been means ± SD and values for ON182′OMe/4LAL/13PS and ON202′OMe/17PS have been in contrast utilizing unpaired two-sided t-test evaluation. ns represents P > 0.05, *represents P < 0.05, **represents P < 0.01, ***represents P < 0.001. Every dot represents one distinct replicate (n = 3). ON312′OMe/17PS scrambled: CCUCAUUCACUCGAUUCA. e Change in cell viability after remedy with 200 nM ONs complexed with LF2000 in comparison with untreated, scale bar = 200 μm. Micrographs of cells proven are the representations from ≥10 photos taken per situation. f Reversed-phase HPLC chromatogram and g. Electrospray (ES-) mass spectrum of purified ON18 used within the assays (required mass = 5976.2, discovered mass = 5975.5). Supply knowledge are supplied within the Supply Information file.

To match organic exercise unbiased of cell uptake, Lipofectamine 2000 (LF2000), a cationic liposome transfection/supply reagent, was used. All three target-complementary PS-ONs have been lively within the assay (ON202′OMe/17PS, ON182′OMe/4LAL/13PS and ON192′OMe/8LNA/17PS), whereas all of the PO-ONs (ON14DNA/4LAL/13PO, ON162′OMe/4LAL/13PO and ON172′OMe/17PO) have been inactive at 100 nM (Fig. 6a). Therefore, in settlement with earlier research, phosphorothioate modification in a target-complementary sequence is important for splice-switching exercise. This might end result from the PS teams enhancing nuclear enrichment53 of the oligonucleotides, and/or recruiting ILF2/3 to the RNA transcript54. Notably, the addition of the amide linkage considerably improved the splice-switching exercise of two′-OMe/PS-ONs on the decrease concentrations (6.25 nM and 12.5 nM), in all probability resulting from improved goal affinity (Fig. 6b). Subsequent, we in contrast the bare (gymnotic) uptake of the ONs. These circumstances extra intently signify in vivo functions the place transfection brokers resembling LF2000 can’t be used. We seeded cells at low confluency, added the oligonucleotides in recent media after 16 h, and measured luciferase exercise after an additional 96 h. The presence of simply 4 LNA-amides (ON182′OMe/4LAL/13PS) considerably elevated exercise in a dose-dependent method in comparison with ON202′OMe/17PS (Fig. 6c). Higher than fivefold improve in exercise was noticed for gymnotic supply in comparison with a most of threefold improve for the LF2000-mediated transfection. This implies that synergy between the PS and LNA-amide modifications results in enhanced productive supply into cells. The improved therapeutic properties of LNA-amide/PS oligonucleotides noticed in these preliminary research are probably resulting from a mixture of diminished cost from the LNA-amides and interactions of the PS spine with mobile parts. Improved cell uptake might end result from the impartial amide linkages breaking apart the poly-anionic spine into brief segments which penetrate cells extra readily than lengthy poly-anionic stretches. Apparently, ON192′OMe/8LNA/PS with LNA and no amides confirmed solely a slight dose-response in exercise, even on the highest focus examined (Fig. 6c and Supplementary Fig. 24). This might be resulting from its binding to off-targets, altered rigidity, or undesirable secondary buildings induced by the acute stability brought on by the LNA sugars, decreasing the flexibility of the ON to work together with the cell floor, a mechanism for productive uptake. ON162′OMe/4LAL/13PO with LNA-amides and no PS linkages additionally displayed slight gymnotic splice-switching exercise (Supplementary Fig. 24).

We in contrast the viability of the HeLa cells following lipofection utilizing a WST-1 cell proliferation assay (Fig. 6d). On the highest focus examined (400 nM) the cells handled with ON202′OMe/17PS have been solely 21% viable, whereas the cells handled with the identical focus of ON182′OMe/4LAL/13PS have been 50% viable, demonstrating that the LNA-amide linkage considerably reduces the cytotoxicity of ONs delivered with LF2000. That is verified by evaluation of the protein ranges (Supplementary Fig. 25) and visual cell dying (Fig. 6e and Supplementary Fig. 26). It helps the usage of the mixture of LNA-amide, 2′-OMe and PS modifications for in vitro research. Apparently, the oligonucleotide containing eight LNA sugars with out amide linkages (ON192′OMe/8LNA/17PS) had a poor toxicity profile. This might be resulting from off-target results and will additionally clarify why, regardless of exhibiting the best affinity in direction of RNA within the UV-melting research, the LNA modified ON192′OMe/8LNA/17PS was not probably the most lively within the exon-skipping assay. Additional detailed research are required to find out whether or not this can be a basic or a sequence-specific phenomenon, and to validate its relevance by way of toxicity.

Provided that cell uptake and toxicity stay main challenges when growing new therapeutic oligonucleotides, the ends in Fig. 6 counsel that our modification technique might be advantageous. The synergistic impact of LNA-amide linkages and phosphorothioate modifications seem to supply oligonucleotides with enhanced organic properties. Nevertheless, cell tradition research can’t deal with among the main challenges in oligonucleotide therapeutics, notably people who relate to pharmacokinetics, pharmacodynamics, biodistribution and facets of toxicity. We’re due to this fact planning additional detailed organic research on LNA-amide-phosphorothioate oligonucleotides to reply these necessary questions.

We have now developed a high-yielding methodology to synthesise oligonucleotides containing uncharged LNA-amide linkages. The chemistry has the potential to be automated and carried out at scale for therapeutic oligonucleotide growth. These new constructs have excessive resistance to enzymatic degradation and bind to complementary RNA with affinity and selectivity superior to unmodified ONs. The synthetic spine causes minimal structural deviation in DNA:RNA hybrids, in keeping with their robust affinity for RNA. Oligonucleotides with alternating LNA-amide and phosphodiester (or phosphorothioate) backbones can’t give rise to recyclable LNA mononucleotides (modified dNTPs) within the presence of mobile nucleases, and their beneficial toxicity profile relative to LNA in these preliminary research might replicate this. In research with gymnotic (bare) supply, combining LNA-amides with phosphorothioates improves cell uptake. Poor mobile uptake is at present a serious barrier in oligonucleotide therapeutics and mixing the PS and LNA modifications with charge-neutral amide backbones resembling AM1 might result in improved scientific efficacy. Analysis is in progress to discover these new analogues in a variety of mobile assays and in different therapeutic interventions resembling siRNA and RNase-H mediated antisense inhibition. Lastly, synthetic nucleic backbones are of curiosity to researchers in lots of different fields together with nucleic acid chemistry, chemical biology, biochemistry, medicinal chemistry, diagnostics, gene synthesis, gene modifying, nanotechnology, supplies chemistry and biophysics. We hope that this work will catalyse future analysis in these areas.

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