Oligonucleotide analogues with locked-amide linkages have therapeutic potential

Oligonucleotide analogues with locked-amide linkages have therapeutic potential | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Oligonucleotide analogues with locked-amide linkages have therapeutic potential Ysobel Baker, Cameron Thorpe, Jinfeng Chen, Laura Poller, Lina Cox, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1542733/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jul, 2022 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Oligonucleotides that target mRNA have great promise as therapeutic agents for life-threatening conditions but suffer from poor bioavailability, hence high cost. As currently untreatable diseases come within the reach of oligonucleotide therapies, new analogues are urgently needed to address this. With this in mind we have developed reduced-charge oligonucleotides containing artificial LNA-amide linkages with improved gymnotic cell uptake, RNA affinity, stability and potency. To construct such oligonucleotides, five LNA-amide monomers (A, T, C, 5mC and G), where the 3´-OH is replaced by an ethanoic acid group, were synthesised in good yield and used in solid-phase oligonucleotide synthesis to form amide linkages with high efficiency. The artificial backbone causes minimal structural deviation to the DNA:RNA duplex. These studies indicate that splice-switching oligonucleotides containing LNA-amide linkages and phosphorothioates display improved activity relative to oligonucleotides lacking amides, highlighting the therapeutic potential of this technology. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Antisense oligonucleotides (ASOs) are synthetic nucleic acid analogues that bind to RNA to regulate gene expression and interfere with protein synthesis 1, 2 . Their modes of action include steric blocking of translation 3 , recruitment of RNase‑H for degradation of mRNA 4 , modulation of pre-mRNA splicing 5-7 and siRNA mediated gene silencing 8 . Therapeutic oligonucleotides in general have shown great promise as precision medicines, and have the potential to cure cancers, genetic disorders, and infectious diseases. They are particularly attractive because there are far more RNA targets than conventional protein targets in currently undruggable diseases 9 . Other advantages include their simple design criteria based on Watson-Crick base pairing and high target specificity. However, although the ASO concept was first demonstrated as far back as 1978 10 , ASOs have only recently started to deliver on their initial promise. Fifteen oligonucleotides have been approved for clinical use (twelve since 2016) indicating their increasing therapeutic value. 11 Until recently all of these were for rare life limiting diseases, but the field just received a major boost with the EMA and NICE approval of the Novartis siRNA drug Inclisiran (Leqvio) for lowering LDL cholesterol levels in patients with hypercholesterolemia 12 . Unlike previous oligonucleotide therapeutics, Inclisiran is used to treat a very common chronic disease. Although a major triumph, and an insight into what the future could hold for oligonucleotide therapeutics, Inclisiran is a special case where targeted delivery to the liver is required. Other organs are more difficult to penetrate, and inefficient biodistribution, poor cellular delivery, and toxicity prevent the wider adoption of antisense technology. Given these limitations, new oligonucleotide analogues are urgently needed to advance the field. To achieve a therapeutic response an oligonucleotide must be stable in vivo , bind to its target RNA with high selectivity and affinity, and display good pharmacokinetic properties 2 . Unmodified oligonucleotides are rapidly digested by nucleases in cells and are therefore completely unsuitable. Modifications designed to provide nuclease resistance 13 include 2´-OMe, 2´- O -(2-methoxyethyl), 2´-fluoro sugars 14-16 , and phosphorothioate (PS) backbones (Fig. 1a) 15, 17 . The PS group improves cell uptake but reduces RNA target affinity, which can be restored by the 2´-sugar substituents 2 . Whilst these modifications have led to oligonucleotide therapies 11 , limited efficacy, off-target effects and toxicity remain obstacles to wider adoption in the clinic 18 . Poor uptake into cells is particularly troublesome; less than 1% of an administered oligonucleotide typically reaches its target 19 . Reducing the net anionic charge of oligonucleotides by partial or complete replacement of the phosphodiester backbone with neutral linkages would seem to be a viable strategy for increasing cell permeability and nuclease resistance 13, 20 . A clinically relevant example 21 is the phosphorodiamidate morpholino backbone (PMO) (Fig. 1a), but limitations in solid phase syntheses complicate the assembly of charge-reduced PMO–DNA chimeras. Most other uncharged backbones that have been studied reduce the affinity and selectivity of therapeutic oligonucleotides towards their RNA targets 13 . However, we felt that the ‘reduced charge’ principle warranted detailed investigation. Oligonucleotides containing the artificial amide backbone AM1 (Fig. 1b) are of interest as they form moderately stable duplexes with complementary RNA 22 23-27 . Despite this, the required monomers have not been synthesised for all four canonical nucleobase analogues 28 . Inspired by the extremely high target affinity of locked nucleic acids (LNA) (Fig. 1b) developed in the 1990’s 29-32 , we hypothesised that conformationally locking the nucleosides surrounding the AM1 linkage would produce reduced-charge oligonucleotides (ONs) with higher target affinity, and in combination with PS linkages might yield ASOs with superior cell uptake and in vivo stability. Although attaching LNA sugars directly to other non-phosphorus-based charge-neutral backbones has so far failed to produce analogues with increased duplex stability 33-38 we reasoned that AM1, being a particularly close analogue of the phosphodiester backbone 39 , might behave differently (Fig. 1c). Here, we describe the synthesis of oligonucleotides containing LNA-amide linkages and show that these can be combined with phosphodiester or phosphorotioate backbones. We demonstrate the excellent duplex stabilisation of the LNA-amide linkages, mismatch discrimination and extreme resistance to nucleases. Preliminary studies show that LNA-amide ONs containing PS backbones have improved cellular uptake and are highly effective in exon-skipping assays. Results And Discussion LNA-amide monomer synthesis is efficient and scalable Efficient synthesis of chemically modified oligonucleotides is essential to fuel fundamental studies and therapeutic applications. We have devised a strategy which enables the straightforward assembly of oligonucleotides containing LNA-flanked amides which could be easily automated and scaled up. A maximum of eight monomers are required to make any sequence that contains non-contiguous amide linkages (four carboxylic acids and four amines). Attaching the required ethanoic acid moiety to the 3´-carbon of the LNA sugar is challenging as it requires removal of the 3´-oxygen and formation of a C-C bond. However, we were able to produce the required 5´-dimethoxytrityl (DMT)-protected 3´-ethanoic acid LNA-monomers in 8 steps from 1 , the same number as for conventional LNA phosphoramidites 43 (Fig. 2a). Moreover, we did this with minimal chromatography. We first built the sugar with the C3´-ester 5 , before addition of the nucleobase. This approach avoids complications associated with forming a C-C bond at the 3´-side of a nucleoside using the Barton-McCombie reaction 23-25, 44 or the hydrogenation step if a Wittig reaction is used 45, 46 , which would ultimately limit the range of heterocyclic bases that can be added. Key intermediate 5 was readily prepared on multiple gram scale in 81% overall yield from commercially available 1 without the need for chromatographic separation as follows. Hydrogenolysis of compound 1 afforded alcohol 2 which was subsequently oxidised to give compound 3 . Olefination of 3 with (carbethoxymethylene)triphenylphosphorane (Wittig reaction) selectively yielded 4 as the ( E )-stereoisomer (Fig. 2b). Catalytic hydrogenation of 4 using Pd/C and H 2 gave 5 as a single stereoisomer (Fig. 2b). This stereoselectivity was predicted because the 1,2- O -isopropylidene groups on the α-face of furanosyl carbohydrate derivatives direct incoming H 2 to the β-face 47 . Key intermediate 5 was then converted to the 1,2-di- O -acetate glycosyl donor 6 following a procedure reported by Arzel et al. 48 , avoiding the formation of a lactone which occurred when the acetonide was cleaved in the presence of water. The pathways to each monomer then diverged, with Vorbrüggen conditions 43, 49 utilised for addition of the nucleobases to access 7a-e . Subsequent simultaneous unmasking of the 3´-carboxyl and 2´-hydroxyl groups by treatment with hydroxide, followed by cyclisation to form the 2´- 4´-oxymethylene bridge, then 5´-mesyl deprotection, gave the hydroxy-LNA acid compounds 8a-e . The progress of the reaction was rapid; mesyl deprotection using hydroxide ion conventionally requires several days under reflux conditions 43 . We postulate that the acceleration in rate is due to neighbouring group participation whereby the carboxylate anion displaces the 5´-mesyl group, forming a lactone that is subsequently opened by hydrolysis (Supplementary Fig. 1). Finally, we treated the resulting hydroxy-acids 8a-e with 4,4´-dimethoxytrityl chloride (DMT-Cl) in pyridine to give the DMT-protected LNA analogue nucleosides 9a-e . Using this strategy we were able to access all four canonical nucleoside analogues along with the 5-methylcytidine monomer which is used in place of cytidine in antisense experiments to increase target affinity and improve other therapeutic properties 18 . Additionally, we required 5´-MMT-amino LNA phosphoramidite 10 . Whilst this had been previously synthesised 40 we chose to develop a more efficient route (Supplementary Fig. 2). Commercially available 5´-MMT-amino dT 11 and 5´-DMT thymidine-3´‑ethanoic acid 12 41, 42 (Fig. 2c) were also required to enable us to make oligonucleotides to compare the properties of DNA amide with those of LNA-amide. Chimeric oligonucleotides can be synthesised in high purity Our oligonucleotide synthesis strategy is shown in Fig. 3. A phosphoramidite monomer with an MMT-protected 5´-amino group, either LNA 10 40 or deoxythymidyl 11 , is added to the oligonucleotide, and the amine is deprotected using trichloroethanoic acid. An LNA-acid (or DNA-acid 50 ) monomer is coupled to the free amine using PyBOP activating agent in the presence of a non-nucleophilic base ( N -methylmorpholine) to form the amide bond. Oligonucleotide synthesis is then resumed, starting with removal of the DMT group. The process is repeated to install multiple non-contiguous amides in the same oligonucleotide. To demonstrate this, DMT-protected LNA acids 9a-e , phosphoramidites 10 and 11 , and DNA acid 12 41, 42 (Fig. 2), were used to synthesise several oligonucleotides, some of which contain multiple additions of LNA-amide, 2´-OMe sugars and PS linkages (Supplementary Table 1). In all cases, we obtained the oligonucleotides in high purity. High-performance liquid chromatography (HPLC) and mass spectrometry demonstrated the high incorporation efficiency for the DMT-protected LNA acids 9a-e (Supplementary Fig. 3-7). LNA sugars stabilise duplexes containing the amide backbone To evaluate the ability of the LNA-amide combination to bind to complementary RNA with high affinity we synthesised a series of 13-mer ONs with a central amide with a T-T sequence. These constructs were composed of either no LNA (ON1 DNA-Am-DNA ), an LNA 5´ to the amide (ON2 LNA-Am-DNA ), an LNA 3´ to the amide (ON3 DNA-Am-LNA ), or LNA on both sides of the amide (ON4 LNA-Am-LNA , Fig. 4a). Controls without amide (ON5 LNA-LNA and ON6 DNAcontrol ) were also made. We compared duplex denaturation temperatures ( T m s) after hybridisation with DNA and RNA complementary strands (Fig. 4b, Supplementary Table 2, Supplementary Fig. 8 and 9). ON2 LNA‑Am-DNA produced a significant increase in DNA:RNA hybrid stability, (+3.0 ˚C) compared to the unmodified ON6 DNAcontrol , and an increase of +3.4˚C compared to ‘amide only’ ON1 DNA-Am-DNA . Importantly, ON4 LNA‑Am-LNA , in which the amide is surrounded by LNA sugars, gave the greatest increase in stability (+5.1 ˚C). It is noteworthy that ON2 LNA-Am-DNA and ON4 LNA-Am-LNA provide the first examples of duplex stabilisation by an LNA sugar attached to a non-phosphorus artificial DNA backbone. The RNA target selectivity of LNA-amide-containing ONs was excellent; a single mismatched base pair greatly reduced duplex stability, in some cases by >14 ˚C (Supplementary Table 2, Supplementary Fig. 10-13) In summary, an amide linkage flanked by LNA on both sides gives strong DNA:RNA duplex stabilisation and good mismatch discrimination. In duplexes with DNA targets, ONs with all combinations of LNA and DNA sugars around the amide linkage were slightly destabilising (between ‑0.1 ˚C to ‑2.6 ˚C), indicating the selectivity of the amide linkage for complementary RNA. The stabilisation induced by the LNA amide combination is cumulative and general (Fig. 4c, Supplementary Table 3, Supplementary Fig. 14-17). In a biologically relevant sequence context, four LNA-amides increase duplex stability against complementary RNA by an impressive 13.0 ˚C compared to only 5.1 ˚C for the DNA target. This large difference is important when developing oligonucleotides to interact with RNA. Oligonucleotides for in vivo studies usually contain 2´-OMe modified sugars and/or phosphorothioate backbones to prevent degradation by nucleases. In such oligonucleotides the combination of LNA and amide also greatly increases duplex stability (Supplementary Table 3). Combining LNA and amide provides strong nuclease resistance Therapeutic oligonucleotides must remain stable in cells for prolonged periods to remain active. To evaluate whether the combination of LNA and amide confers greater nuclease resistance than LNA alone, we incubated unmodified DNA (ON15 DNA/17PO ) and DNA with four LNA-amide linkages (ON14 DNA/4LAL/13PO ) in a 1:1 mixture of phosphate buffered saline (PBS) and foetal bovine serum (FBS) to mimic the in vivo environment, and compared it to the equivalent construct with LNA but without amide linkages (ON25 DNA/8LNA/17PO , Supplementary Fig. 18). The results show that the combination of LNA and amide confers extreme resistance to nucleases. Both the oligonucleotides lacking amide linkages had partially degraded within 1 hour whereas ON14 DNA/4LAL/13PO remained intact after 8 hours. Interestingly ON14 shows stability at its 3´-end, even though this region has an unmodified sugar phosphate trimer. The 3´-terminal pentamer region, however, is highly modified. It has reduced charge due to the amide linkage and also contains two LNA sugars. It may therefore not be recognised by nucleases. This enhanced stability further illustrates the advantages of removing charge and including modified sugars in antisense oligonucleotides. X-ray crystallography of the LNA-amide modification We solved several X-ray structures to determine the effects of LNA and amide modifications on duplex structure and conformation. These are the first crystal structures of DNA:RNA hybrids that contain amide linkages. An amide-modified RNA:RNA duplex was analysed previously, but this had amides in both strands surrounded by multiple mismatched base pairs which cannot exist outside the solid-state at ambient temperature 27 . The sequence of the modified DNA:RNA hybrid duplexes (Supplementary Table 4) was based on the corresponding unmodified version (d-CT TT TCTTTG/rCAAAGAAAAG) 51 (the location of the amide is underlined). Good quality crystals diffracting between 2.5-2.8 Å resolution were obtained for the DNA:RNA hybrid in which the DNA strand contains an amide linkage flanked by DNA on both sides, LNA on both sides and with LNA only on the 5´-side (Supplementary Fig. 19). The unmodified DNA:RNA duplex was also studied. The data collection and refinement statistics are given in Supplementary Table 5. Electron density maps at the modification position for the four nucleic acid crystal structures reported in this manuscript are given in Supplementary Fig. 20. The hybrids with amide and LNA-amide backbones (Fig. 5a) are structurally very similar to the unmodified duplex (all-atom RMSD 0.4 Å) as shown by their superimposition (Fig. 5a, Supplementary Fig. 21). All structures adopt the A-conformation with sugar puckers clustering around C3´- endo (Supplementary Fig. 22). As expected, all duplexes are stabilised by canonical Watson-Crick base pairs, indicating that the thermodynamic improvements due to the LNA-amide backbones are not due to unusual changes in hydrogen bonding interactions. In agreement with the DNA:RNA hybrid NMR structure by Rosners 22 in which the DNA strand contained multiple amides, our X-ray studies indicate that the amide linkage is a close mimic of the phosphodiester backbone (Fig. 5b). Both are four-atom linkages, hence similar in length, and the amide carbonyl is orientated in the same direction as one of the phosphodiester P‑O bonds. In Fig. 5c, the structures of all amide backbones are overlaid to assess the effects of the LNA modifications. Between each structure, the orientation of the backbone is consistent, directing the amide oxygen into the major groove. Other atomic positions of the backbones also show close similarity, and the presence of 3´-LNA causes no significant distortion. 5´-LNA does however cause some structural displacement; the 5´-sugars in the LNA-amide-DNA and LNA-amide-LNA structures are shifted slightly outwards compared to the DNA-amide-DNA and unmodified strands. Despite this, the positioning of the amide backbone remains consistent between each structure. The amide adopts the expected trans- conformation, and LNA on the 5´-side of the amide has little effect on backbone torsion angles (Fig. 5c, Supplementary Fig. 23). In summary, combined LNA and amide modifications have minimal effect on the duplex structure, and are excellent mimics of natural phosphodiesters. Combining LNA-amide and PS enhances gymnotic delivery In a preliminary study we have evaluated the biological activity of the LNA-amide combination using the HeLa pLuc/705 cell line 52 that carries a luciferase-encoding gene interrupted by a mutated ß-globin intron 52 . This mutation creates a 5´-splice site which activates a cryptic 3´-splice site, resulting in incorrect mRNA splicing and the production of non-functional luciferase. An oligonucleotide that hybridises to the mutant 5´-splice site prevents incorporation of the aberrant intron, restoring the pre-mRNA splicing to produce functional luciferase, which is quantified by luminometry. Oligonucleotides complementary to this aberrant splice site were synthesised with combinations of different modifications to determine their individual effects (Supplementary Table 3). ON14 DNA/4LAL/13PO , ON16 2 ¢ OMe/4LAL/13PO , and ON18 2 ¢ OMe/4LAL/13PS were designed to evaluate LNA-amide with the DNA, 2´‑OMe/phosphodiester, and 2´-OMe/phosphorothioate backbones respectively, and ONs 14 and 16 were included to determine the degree to which LNA-amide influences delivery, activity and toxicity in the absence of PS linkages. LNA and amide linkages are incompatible with RNase-H so there is no risk of ON14 or ON16 inadvertently destroying the RNA target 2,56 . Three controls were included: ON20 2 ¢ OMe/17PS (which represents the gold standard in the assay) to determine whether LNA-amide enhances biological activity 52 , ON17 2 ¢ OMe/17PO to evaluate the effects of the PS linkage independently of LNA or amide linkages, and ON19 2 ¢ OMe/8LNA/17PS with LNA sugars but no amide linkages to determine the effects of the enhanced duplex stability caused by LNA. A scrambled control with a 2´-OMe/PS backbone was also included to determine off target effects (ON31 2 ¢ OMe/17PS scrambled , Fig. 6). To compare biological activity independent of cell uptake, Lipofectamine 2000 (LF2000), a cationic liposome transfection/delivery reagent, was used. All three target-complementary PS-ONs were active in the assay (ON20 2 ¢ OMe/17PS , ON18 2 ¢ OMe/4LAL/13PS , and ON19 2 ¢ OMe/8LNA/17PS ), whereas all the PO-ONs (ON14 DNA/4LAL/13PO , ON16 2 ¢ OMe/4LAL/13PO and ON17 2 ¢ OMe/17PO ) were inactive at 100 nM (Fig. 6a). Hence, in agreement with previous studies, the phosphorothioate modification in a target-complementary sequence is necessary for splice-switching activity. This could result from the PS groups enhancing nuclear enrichment 53 of the oligonucleotides, and/or recruiting ILF2/3 to the RNA transcript 54 . Notably, the addition of the amide linkage significantly improved the splice-switching activity of 2´-OMe/PS ONs at the lower concentrations (6.25 nM and 12.5 nM), probably due to improved target affinity (Fig. 6b). Next, we compared the naked (gymnotic) uptake of the ONs. These conditions more closely represent in vivo applications where transfection agents such as LF2000 cannot be used. We seeded cells at low confluency, added the oligonucleotides in fresh media after 16 h, and measured luciferase activity after a further 96 h. The presence of just four LNA-amides (ON18 2 ¢ OMe/4LAL/13PS ) significantly increased activity in a dose-dependent manner compared to ON20 2 ¢ OMe/17PS (Fig. 6c). Greater than 5-fold increase in activity was observed for gymnotic delivery compared to a maximum of 3-fold increase for the LF2000‑mediated transfection. This suggests that synergy between the PS and LNA-amide modifications leads to enhanced productive delivery into cells. The improved therapeutic properties of LNA-amide/PS oligonucleotides observed in these preliminary studies are possibly due to a combination of reduced charge from the LNA-amides and interactions of the PS backbone with cellular components. Improved cell uptake may result from the neutral amide linkages breaking up the poly-anionic backbone into short segments which penetrate cells more readily than long poly-anionic stretches. Interestingly, ON19 2 ¢ OMe/8LNA/PS with LNA and no amides showed only slight dose response in activity, even at the highest concentration tested (Fig. 6c, Supplementary Fig. 24). This could be due to its binding to off-targets, altered rigidity, or undesirable secondary structures induced by the extreme stability caused by the LNA sugars, reducing the ability of the ON to interact with the cell surface, a mechanism for productive uptake. ON16 2 ¢ OMe/4LAL/13PO with LNA-amides and no PS linkages also displayed slight gymnotic splice-switching activity (Supplementary Fig. 24). We compared the viability of the HeLa cells following lipofection using a WST-1 cell proliferation assay (Fig. 6d). At the highest concentration tested (400 nM) the cells treated with ON20 2 ¢ OMe/17PS were only 21% viable, whereas the cells treated with the same concentration of ON18 2 ¢ OMe/4LAL/13PS were 50% viable, demonstrating that the LNA-amide linkage significantly reduces the cytotoxicity of ONs delivered with LF2000. This is verified by analysis of the protein levels (Supplementary Fig. 25) and visible cell death (Fig. 6e, and Supplementary Fig. 26). It supports the use of the combination of LNA-amide, 2´-OMe and PS modifications for in-vitro studies. Interestingly, the oligonucleotide containing eight LNA sugars without amide linkages (ON19 2 ¢ OMe/8LNA/17PS ) had a poor toxicity profile. This could be due to off-target effects and could also explain why, despite showing the highest affinity towards RNA in the UV melting studies, the LNA modified ON19 2 ¢ OMe/8LNA/17PS was not the most active in the exon-skipping assay. Further detailed studies are required to determine whether this is a general or a sequence specific phenomenon, and to validate its relevance in terms of toxicity. Given that cell uptake and toxicity remain major challenges when developing new therapeutic oligonucleotides the results in Fig. 6 suggest that our modification strategy could be advantageous. The synergistic effect of LNA-amide linkages and phosphorothioate modifications appear to produce oligonucleotides with enhanced biological properties. However, cell culture studies cannot address some of the major challenges in oligonucleotide therapeutics, particularly those that relate to pharmacokinetics, pharmacodynamics, biodistribution and aspects of toxicity. We are therefore planning further detailed biological studies on LNA-amide-phosphorothioate oligonucleotides to answer these important questions. Conclusions We have 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 development. These new constructs have high resistance to enzymatic degradation and bind to complementary RNA with affinity and selectivity superior to unmodified ONs. The artificial backbone causes minimal structural deviation in DNA:RNA hybrids, consistent with their strong affinity for RNA. Oligonucleotides with alternating LNA-amide and phosphodiester (or phosphorothioate) backbones cannot give rise to recyclable LNA mononucleotides (modified dNTPs) in the presence of cellular nucleases, and their favourable toxicity profile relative to LNA in these initial studies may reflect this. In studies with gymnotic (naked) delivery, combining LNA-amides with phosphorothioates improves cell uptake. Poor cellular uptake is currently a major barrier in oligonucleotide therapeutics and combining the PS and LNA modifications with charge-neutral amide backbones such as AM1 could lead to improved clinical efficacy. Research is in progress to explore these new analogues in a range of cellular assays and in other therapeutic interventions such as siRNA and RNase-H mediated antisense inhibition. Finally, artificial nucleic backbones are of interest to researchers in many other fields including nucleic acid chemistry, chemical biology, biochemistry, medicinal chemistry, diagnostics, gene synthesis, gene editing, nanotechnology, materials chemistry and biophysics. We hope that this work will catalyse future research in these areas. Methods Amide-modified oligonucleotide synthesis Oligonucleotide segment synthesis. Oligonucleotide synthesis was performed on an Applied Biosystems 394 automated DNA/RNA synthesiser on a 1.0 µmole scale using a standard phosphoramidite cycle of detritylation, coupling, and oxidation. No capping step was used. All β-cyanoethyl phosphoramidite monomers were dissolved in anhydrous MeCN (10% CH 2 Cl 2 was added when 2′-OMe U phosphoramidite was used) to a concentration of 0.1 M immediately prior to use. 5-(Benzylthio)-1 H -tetrazole (BTT) activator (0.3 M) was used with a coupling time of 50 s for normal dA, dG, dC and T phosphoramidites, this was extended to 6 min for 2′-OMe and LNA phosphoramidites. Standard iodine oxidiser was used for phosphodiester oligonucleotides. For the phosphorothioate oligonucleotides, 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) was used as the sulfurisation agent, and the solid support was washed with MeCN after each phosphoramidite coupling before the sulfurisation step. Sulfurisation time was initially 3 min, and after this period fresh EDITH was sent to the synthesis column and left for another 3 min. Further details are given in the Supplementary Information. Amino monomer addition. The MMT-protected 5´-amino phosphoramidite monomer (either LNA 10 40 or commercially available deoxythymidyl 11 ) was dissolved in anhydrous MeCN to a concentration of 0.1 M immediately prior to use. The same coupling conditions as above were used, but the coupling time was extended to 10 min. The MMT protecting group was cleaved on the Applied Biosystems 394 automated synthesiser using 3% TCA in CH 2 Cl 2 with an extended cleavage time of 2 min. The solid support was then washed with acetonitrile on the synthesiser for 3 min. To improve the coupling efficiency in the next step, the solid support was washed with 0.5% (v/v) N -methylmorpholine in DMF (1 x 1mL) followed by DMF (3 x 1 mL). Amide bond formation on resin (peptide coupling). All amide couplings were performed manually on the synthesis column. A solution with 10 equivalents of acid monomer, 10 equivalents of PyBOP and 30 equivalents of N -methylmorpholine was first prepared in 400 µL of DMF. This was then taken up into a 1 mL syringe and loaded onto the column before a second 1 mL syringe was attached to the other end of the synthesis column. The mixture was agitated every 10 min for 1 h. The columns were then washed with DMF (3 x 1 mL) followed by MeCN (5 x 1 mL) and dried by passing argon through the column. The column was then returned to the synthesiser to continue oligonucleotide synthesis. Cleavage of oligonucleotides from resin, deprotection and purification. LNA-amide containing oligonucleotides were isolated with the final 5′-DMT protecting group still in place (DMT-ON). Following solid phase synthesis, the cyanoethyl groups were removed by a 15 min treatment with 20% diethylamine in MeCN. The resin was then washed with MeCN (5 x 1 mL) and dried by passing a stream of argon through the synthesis column. The oligonucleotides were cleaved from the solid support and deprotected by heating in concentrated aqueous ammonia in a sealed glass vial at 55 °C for 5 hours. The ammonia was removed under reduced pressure prior to oligonucleotide purification. The DMT-ON oligonucleotides were purified by reverse-phase high performance liquid chromatography (RP-HPLC) and lyophilised. They were then dissolved in 0.5 mL of 80% acetic acid and left for 1 hour at room temperature to remove the DMT group. The solution was neutralised with 0.5 mL of triethylammonium acetate buffer (2 M, pH 7) and the detritylated oligonucleotides were desalted using a NAP-10 column (Cytiva) then freeze dried. Data availability Crystallographic data for the structures in this Article have been deposited at the Cambridge Crystallographic Data Centre under deposition numbers CCDC 2105684 and 2105685.DNA structural data obtained by X-ray crystallography were deposited in the Protein Data Bank (PDB) and are available with the following accession codes 7NRP, 7OOS, 7OZZ and 7OOO. Source data is provided for Fig. 6 and Supplementary Figs. 24 and 25. The authors declare that all other data supporting the findings of this study are available within the paper and its supplementary information files. Declarations Acknowledgements The authors would like to thank Dr Samir El-Andaloussi for the HeLa pLuc/705 cell line 52 . This work was funded in part by a BBSRC research grant to TB (New oligonucleotide analogues for therapeutic applications. BB/S018794/1). Author contributions Y.R.B, L.M.P, L.L and P.K performed the small molecule synthesis; Y.R.B, L.M.P and K.E.C carried out the small molecule crystallography work; Y.R.B, A.E.S, J.C, S.E and D.S synthesised and purified the ONs; Y.R.B, J.C and L.C performed UV melting and enzymatic stability studies; C.T, M.A.McD, J.S.H and J.P.H carried out the ON XRD studies; Y.R.B, W.F.L and G.McC performed the cell studies; Y.R.B, A.E.S, P.K and T.B. designed the study. All authors contributed to the writing of the manuscript. T.B. oversaw and managed the project. M.J.A.W oversaw the biological work. Competing interests T.B.is a consultant to ATDBio Ltd. References Smith, C.I.E. & Zain, R. Therapeutic Oligonucleotides: State of the Art. Annu. Rev. Pharmacol. Toxicol. 59 , 605–630 (2019). Khvorova, A. & Watts, J.K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol. 35 , 238–248 (2017). Rinaldi, C. & Wood, M.J.A. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nature Reviews Neurology 14 , 9–21 (2018). Liang, X.-H., Sun, H., Nichols, J.G. & Crooke, S.T. RNase H1-Dependent Antisense Oligonucleotides Are Robustly Active in Directing RNA Cleavage in Both the Cytoplasm and the Nucleus. Mol. Ther. 25 , 2075–2092 (2017). Dominski, Z. & Kole, R. Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides. Proc. Natl. Acad. Sci. U. S. A. 90 , 8673–8677 (1993). Sierakowska, H., Sambade, M.J., Agrawal, S. & Kole, R. Repair of thalassemic human beta-globin mRNA in mammalian cells by antisense oligonucleotides. Proc. Natl. Acad. Sci. U. S. A. 93 , 12840–12844 (1996). Aartsma-Rus, A. et al. Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum. Mol. Genet. 12 , 907–914 (2003). Setten, R.L., Rossi, J.J. & Han, S.-p. The current state and future directions of RNAi-based therapeutics. Nature Reviews Drug Discovery 18 , 421–446 (2019). Warner, K.D., Hajdin, C.E. & Weeks, K.M. Principles for targeting RNA with drug-like small molecules. Nature Reviews Drug Discovery 17 , 547–558 (2018). Zamecnik, P.C. & Stephenson, M.L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. U. S. A. 75 , 280–284 (1978). Xiong, H., Veedu, R.N. & Diermeier, S.D. Recent Advances in Oligonucleotide Therapeutics in Oncology. Int. J. Mol. Sci. 22 , 3295 (2021). Fitzgerald, K. et al. A Highly Durable RNAi Therapeutic Inhibitor of PCSK9. N. Engl. J. Med. 376 , 41–51 (2016). Freier, S.M. & Altmann, K.-H. The ups and downs of nucleic acid duplex stability: Structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 25 , 4429–4443 (1997). Cummins, L.L. et al. Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity. Nucleic Acids Res. 23 , 2019–2024 (1995). Monia, B.P., Johnston, J.F., Sasmor, H. & Cummins, L.L. Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras. J. Biol. Chem. 271 , 14533–14540 (1996). Martin, P. A New Access to 2'-O-Alkylated Ribonucleosides and Properties of 2'-O-Alkylated Oligoribonucleotides. Helv. Chim. Acta 78 , 486–504 (1995). Eckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. 24 , 374–387 (2014). Roberts, T.C., Langer, R. & Wood, M.J.A. Advances in oligonucleotide drug delivery. Nature Reviews Drug Discovery 19 , 673–694 (2020). Godfrey, C. et al. Delivery is key: lessons learnt from developing splice-switching antisense therapies. EMBO Mol. Med. 9 , 545–557 (2017). Meade, B.R. et al. Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications. Nat. Biotechnol. 32 , 1256–1261 (2014). Aartsma-Rus, A. & Krieg, A.M. FDA Approves Eteplirsen for Duchenne Muscular Dystrophy: The Next Chapter in the Eteplirsen Saga. Nucleic Acid Ther. 27 , 1–3 (2016). Selvam, C., Thomas, S., Abbott, J., Kennedy, S.D. & Rozners, E. Amides as Excellent Mimics of Phosphate Linkages in RNA. Angew. Chem. Int. Ed. 50 , 2068–2070 (2011). Idziak, I., Just, G., Damha, M.J. & Giannaris, P.A. Synthesis and hybridization properties of amide-linked thymidine dimers incorporated into oligodeoxynucleotides. Tetrahedron Lett. 34 , 5417–5420 (1993). Lebreton, J., Waldner, A., Lesueur, C. & De Mesmaeker, A. Antisense Oligonucleotides with Alternating Phosphodiester-"Amide-3" Linkages. Synlett 1994, 137–140 (1994). De Mesmaeker, A. et al. Amides as a New Type of Backbone Modification in Oligonucleotides. Angew. Chem. Int. Ed. 33 , 226–229 (1994). Hardcastle, T. et al. A Single Amide Linkage in the Passenger Strand Suppresses Its Activity and Enhances Guide Strand Targeting of siRNAs. ACS Chem. Biol. 13 , 533–536 (2018). Mutisya, D. et al. Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs. Nucleic Acids Res. 45 , 8142–8155 (2017). Kotikam, V. & Rozners, E. Amide-Modified RNA: Using Protein Backbone to Modulate Function of Short Interfering RNAs. Acc. Chem. Res. 53 , 1782–1790 (2020). Hagedorn, P.H. et al. Locked nucleic acid: modality, diversity, and drug discovery. Drug Discovery Today 23 , 101–114 (2018). Singh, S.K., A. Koshkin, A., Wengel, J. & Nielsen, P. LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem. Commun., 455–456 (1998). Singh, S.K. & Wengel, J. Universality of LNA-mediated high-affinity nucleic acid recognition. Chem. Commun., 1247–1248 (1998). Bondensgaard, K. et al. Structural Studies of LNA:RNA Duplexes by NMR: Conformations and Implications for RNase H Activity. Chem. Eur. J. 6 , 2687–2695 (2000). Sharma, V.K. et al. Synthesis and biological properties of triazole-linked locked nucleic acid. Chem. Commun. 53 , 8906–8909 (2017). Kumar, P., Truong, L., Baker, Y.R., El-Sagheer, A.H. & Brown, T. Synthesis, Affinity for Complementary RNA and DNA, and Enzymatic Stability of Triazole-Linked Locked Nucleic Acids (t-LNAs). ACS Omega 3 , 6976–6987 (2018). Kumar, P., El-Sagheer, A.H., Truong, L. & Brown, T. Locked nucleic acid (LNA) enhances binding affinity of triazole-linked DNA towards RNA. Chem. Commun. 53 , 8910–8913 (2017). Thorpe, C., Epple, S., Woods, B., El-Sagheer, A.H. & Brown, T. Synthesis and biophysical properties of carbamate-locked nucleic acid (LNA) oligonucleotides with potential antisense applications. Org. Biomol. Chem. 17 , 5341–5348 (2019). Lauritsen, A. & Wengel, J. Oligodeoxynucleotides containing amide-linked LNA-type dinucleotides: synthesis and high-affinity nucleic acid hybridization. Chem. Commun., 530–531 (2002). Seth, P.P. & Swayze, E.E. Oligomeric compounds comprising neutrally linked terminal bicyclic nucleosides PCT/US2009/039438 ISIS Pharmaceuticals WO/2009/124238 2009 Shivalingam, A., Tyburn, A.E.S., El-Sagheer, A.H. & Brown, T. Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation. J. Am. Chem. Soc. 139 , 1575–1583 (2017). Obika, S., Nakagawa, O., Hiroto, A., Hari, Y. & Imanishi, T. Synthesis and properties of a novel bridged nucleic acid with a P3′ → N5′ phosphoramidate linkage, 5′-amino-2′,4′-BNA. Chem. Commun., 2202–2203 (2003). Stork, G., Zhang, C., Gryaznov, S. & Schultz, R. Modified ollgonucleotides. Effect of 4 vs 5-atom chimeric internucleoside linkages on duplex stability. Tetrahedron Lett. 36 , 6387–6390 (1995). Ammenn, J., Altmann, K.-H. & Belluš, D. Aza-Claisen Rearrangement: Synthesis of 5′-branched 5′-aminothymidines. Helv. Chim. Acta 80 , 1589–1606 (1997). Koshkin, A.A., Fensholdt, J., Pfundheller, H.M. & Lomholt, C. A Simplified and Efficient Route to 2′-O, 4′-C-Methylene-Linked Bicyclic Ribonucleosides (Locked Nucleic Acid). J. Org. Chem. 66 , 8504–8512 (2001). Lebreton, J., Waldner, A., Fritsch, V., Wolf, R.M. & De Mesmaeker, A. Comparison of two amides as backbone replacement of the phosphodiester linkage in oligodeoxynucleotides. Tetrahedron Lett. 35 , 5225–5228 (1994). Peterson, M.A. et al. Amide-Linked Ribonucleoside Dimers Derived from 5′-Amino-5′-deoxy- and 3′-(Carboxymethyl)-3′-deoxynucleoside Precursors(1). J. Org. Chem. 64 , 8183–8192 (1999). Kotikam, V. & Rozners, E. Concurrent Hydrogenation of Three Functional Groups Enables Synthesis of C3′-Homologated Nucleoside Amino Acids. Org. Lett. 19 , 4122–4125 (2017). Albrecht, H., Jones, G. & Moffatt, J. 3'-Deoxy-3'-(dihydroxyphosphinylmethyl)nucleosides. Isophosphonate Analogs of Nucleoside 3'-Phosphates. J. Amer. Chem. Soc. 92 , 5511–5513 (1970). Arzel, L. et al. Synthesis of Ribonucleosidic Dimers with an Amide Linkage from d-Xylose. J. Org. Chem. 81 , 10742–10758 (2016). Vorbrüggen, H. Some recent trends and progress in nucleoside synthesis. Acta Biochim. Pol. 43 , 25–36 (1996). Brandsen, B.M., Hesser, A.R., Castner, M.A., Chandra, M. & Silverman, S.K. DNA-Catalyzed Hydrolysis of Esters and Aromatic Amides. J. Amer. Chem. Soc. 135 , 16014–16017 (2013). Kopka, M.L., Lavelle, L., Han, G.W., Ng, H.-L. & Dickerson, R.E. An Unusual Sugar Conformation in the Structure of an RNA/DNA Decamer of the Polypurine Tract May Affect Recognition by RNase H. J. Mol. Biol. 334 , 653–665 (2003). Kang, S.-H., Cho, M.-J. & Kole, R. Up-Regulation of Luciferase Gene Expression with Antisense Oligonucleotides: Implications and Applications in Functional Assay Development. Biochemistry 37 , 6235–6239 (1998). Bailey, J.K., Shen, W., Liang, X.-H. & Crooke, S.T. Nucleic acid binding proteins affect the subcellular distribution of phosphorothioate antisense oligonucleotides. Nucleic Acids Res. 45 , 10649–10671 (2017). Rigo, F. et al. Synthetic oligonucleotides recruit ILF2/3 to RNA transcripts to modulate splicing. Nat. Chem. Biol. 8 , 555–561 (2012). Additional Declarations Yes there is potential Competing Interest. T.B.is a consultant to ATDBio Ltd. Supplementary Files Sourcedata.xlsx source data for Figure 6 and Supplementary Figure 25 supportinginformationforNatCommunrevised.pdf Supporting information RS.pdf Reporting Summary Onlinefloatimage1.png Graphical Abstract Cite Share Download PDF Status: Published Journal Publication published 12 Jul, 2022 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1542733","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":98715828,"identity":"9f5bbe90-8d1f-47f3-a048-b170748dcabf","order_by":0,"name":"Ysobel Baker","email":"","orcid":"https://orcid.org/0000-0002-0266-771X","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Ysobel","middleName":"","lastName":"Baker","suffix":""},{"id":98715829,"identity":"eea4b38d-70ae-48d0-b344-eb88d43cc6f4","order_by":1,"name":"Cameron Thorpe","email":"","orcid":"https://orcid.org/0000-0002-1980-678X","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Cameron","middleName":"","lastName":"Thorpe","suffix":""},{"id":98715830,"identity":"7454b901-06d0-48e0-9c33-3974ead2dded","order_by":2,"name":"Jinfeng Chen","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Jinfeng","middleName":"","lastName":"Chen","suffix":""},{"id":98715831,"identity":"91de344d-33b2-4f98-a289-510c53e4dfb0","order_by":3,"name":"Laura Poller","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Poller","suffix":""},{"id":98715832,"identity":"123c1dfd-9d29-4ddc-bd92-2f44ce33f2d3","order_by":4,"name":"Lina Cox","email":"","orcid":"https://orcid.org/0000-0001-9450-6819","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Lina","middleName":"","lastName":"Cox","suffix":""},{"id":98715833,"identity":"670787c4-d3cd-48ba-b68a-7c88f21cfc83","order_by":5,"name":"Pawan Kumar","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Pawan","middleName":"","lastName":"Kumar","suffix":""},{"id":98715834,"identity":"dece844f-bdf8-4fb4-ae6e-b8da5db56786","order_by":6,"name":"Wooi Fang Lim","email":"","orcid":"https://orcid.org/0000-0002-1238-3040","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Wooi","middleName":"Fang","lastName":"Lim","suffix":""},{"id":98715835,"identity":"e3b0baff-006d-40df-b7d1-87054947f0e5","order_by":7,"name":"Lillian Lie","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Lillian","middleName":"","lastName":"Lie","suffix":""},{"id":98715836,"identity":"1c20e209-6073-441c-a3a9-174f0be31374","order_by":8,"name":"Graham McClorey","email":"","orcid":"","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Graham","middleName":"","lastName":"McClorey","suffix":""},{"id":98715837,"identity":"3f50f783-4b48-4bcf-833d-baa033500545","order_by":9,"name":"Sven Epple","email":"","orcid":"https://orcid.org/0000-0002-9078-3250","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Sven","middleName":"","lastName":"Epple","suffix":""},{"id":98715838,"identity":"9fd2fc66-ca06-45e4-939f-d4891699912a","order_by":10,"name":"Daniel Singleton","email":"","orcid":"","institution":"ATDBio","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Singleton","suffix":""},{"id":98715839,"identity":"e8c789c3-c7ff-4d7e-a832-dab8f668a19a","order_by":11,"name":"Michael McDonough","email":"","orcid":"https://orcid.org/0000-0003-4664-6942","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"McDonough","suffix":""},{"id":98715840,"identity":"3bc3eac5-c127-469f-9bc2-16afd4603dc7","order_by":12,"name":"Jack Hardwick","email":"","orcid":"https://orcid.org/0000-0002-3117-1035","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Jack","middleName":"","lastName":"Hardwick","suffix":""},{"id":98715841,"identity":"8259defe-be74-418c-9817-2a4e92ddbe14","order_by":13,"name":"Kirsten Christensen","email":"","orcid":"https://orcid.org/0000-0003-1683-2066","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Kirsten","middleName":"","lastName":"Christensen","suffix":""},{"id":98715842,"identity":"81a743ce-ebfd-412b-9b95-b74ddae0f71c","order_by":14,"name":"Matthew Wood","email":"","orcid":"https://orcid.org/0000-0002-5436-6011","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Wood","suffix":""},{"id":98715843,"identity":"f2b371a6-a90f-42f0-b497-529ba70cc59e","order_by":15,"name":"James Hall","email":"","orcid":"https://orcid.org/0000-0003-3716-4378","institution":"University of Reading","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"","lastName":"Hall","suffix":""},{"id":98715844,"identity":"dd5e1fca-dbaa-4b91-97d6-a8d5a20e6d12","order_by":16,"name":"Afaf El-Sagheer","email":"","orcid":"https://orcid.org/0000-0001-8706-1292","institution":"University of Oxford","correspondingAuthor":false,"prefix":"","firstName":"Afaf","middleName":"","lastName":"El-Sagheer","suffix":""},{"id":98715845,"identity":"f9640da1-2a19-4cbd-b19c-9ce5a415cf33","order_by":17,"name":"Tom Brown","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYJACZjDJ3sCGLJiAUzkPXAvPAZK1SCQQqcWevTuBuXCPTeJ2ycfPHnzMYZAzOMD88ANjWxpuW3jObmCe8SwtcefsNHPDmdsYjCUb2IwlGNtycGuRyN3AzHPgcO6G2zls0rzbGBL7GRjMGBjbKojQcvMMm/RfoJY2BvZvRGq5wcMmzQi2hQdkCx6HnTm74fCMA2n1G86kmUn2bpMwlmzmKZZIOIfb++ztvRsfFxywMTY4fviZxM9tNnIGx9s3fvhQloxTCwgcQGJLQKIpAa+GUTAKRsEoGAWEAACIG02Kng6QoQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-6538-3036","institution":"University of Oxford","correspondingAuthor":true,"prefix":"","firstName":"Tom","middleName":"","lastName":"Brown","suffix":""}],"badges":[],"createdAt":"2022-04-10 12:00:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1542733/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1542733/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-022-31636-2","type":"published","date":"2022-07-12T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":21082004,"identity":"2c477358-2bbd-48b8-97b2-a0fb28109961","added_by":"auto","created_at":"2022-05-04 19:03:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTherapeutic oligonucleotide modifications and our strategy for combining these. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ea. \u003c/em\u003e\u003c/strong\u003e\u003cem\u003eExisting therapeutic oligonucleotide modifications and their properties. Traffic light coding system: green plus = good; amber circle = intermediate; red cross = poor. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eb. \u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;Oligonucleotide modifications that have not yet reached the clinic \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ec.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Development of a new antisense backbone chemistry with improved target affinity and cellular activity. The LNA-amide linkage is achiral, and can be prepared by solid phase synthesis methods that are high yielding with potential to be automated.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/4b42dc89abdffeb8db586cd7.png"},{"id":21081780,"identity":"3036ccdc-d2fa-491d-95b0-f2631310243b","added_by":"auto","created_at":"2022-05-04 18:58:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":184172,"visible":true,"origin":"","legend":"\u003cp\u003eLNA-ethanoic acids and other monomers used in this study. \u003cstrong\u003ea.\u003c/strong\u003e Synthesis of the DMT-protected LNA ethanoic acids \u003cstrong\u003e9a-e\u003c/strong\u003e. \u003cstrong\u003eb\u003c/strong\u003e. X-ray crystal structures confirming the (E)-configuration in \u003cstrong\u003e4\u003c/strong\u003e and the stereochemistry at the 3´-carbon in \u003cstrong\u003e5 \u003c/strong\u003e(details in the Supplementary Information). \u003cstrong\u003ec\u003c/strong\u003e. Phosphoramidites \u003cstrong\u003e10\u003c/strong\u003e\u003csup\u003e40\u003c/sup\u003e\u003cstrong\u003e \u003c/strong\u003eand \u003cstrong\u003e11 \u003c/strong\u003e(commercially available) and \u0026nbsp;DNA-ethanoic acid monomer \u003cstrong\u003e12\u003c/strong\u003e\u003csup\u003e41, 42\u003c/sup\u003e used to synthesise oligonucleotides. \u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/b1a21ef19afad2f6cbf5910a.png"},{"id":21081776,"identity":"1c16c01b-ec9f-43aa-b3aa-222304da76a9","added_by":"auto","created_at":"2022-05-04 18:58:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":74192,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003ea.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Efficient solid-phase synthesis of amide-phosphodiester chimeras. Dashed lines indicate presence or absence of 2´-4´-methylene bridge. PyBOP = (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eb.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Reversed-phase HPLC chromatogram and \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ec.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Electrospray (ES-) mass spectrum of a crude LNA-amide chimeric oligonucleotide (ON14). (required mass = 5438.7, found mass = 5439.0).\u0026nbsp;\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/08b7b2bdccca835a22f93426.png"},{"id":21082002,"identity":"72ae5ec9-ea77-4bb6-8ca7-ee13d0e76c08","added_by":"auto","created_at":"2022-05-04 19:03:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33765,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCombining LNA and AM1 increases affinity for complementary RNA. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ea.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e The structures of each of the LNA AM1 combinations studied. Sequence d-CGACGC\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eTT\u003c/u\u003e\u003c/em\u003e\u003c/strong\u003e\u003cem\u003eGCAGC\u003c/em\u003e \u003cem\u003e(where the underlined region indicates the modification position)\u003c/em\u003e \u003cstrong\u003e\u003cem\u003eb.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e UV-melting curve derivatives of the ONs from panel \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ea\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e in duplexes with complementary RNA. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ec. \u003c/em\u003e\u003c/strong\u003e\u003cem\u003eUV-melting curve derivatives demonstrating that the stabilisation conferred by the LNA-amide linkage against complementary RNA is general and cumulative. Conditions: 10 mM phosphate buffer, 200 mM NaCl, pH 7.0. Sequences in Supplementary Table 1.\u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/22a6956bc8ad0cc4c1f16fd6.png"},{"id":21081774,"identity":"1681f5fc-d584-46ad-8270-0ce769b3fcf6","added_by":"auto","created_at":"2022-05-04 18:58:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":227194,"visible":true,"origin":"","legend":"\u003cp\u003eX-ray structures of amide and LNA-amide modified DNA:RNA duplexes. \u003cstrong\u003ea\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eStructural identity of amide and LNA-amide modifications and the torsion angles of the amide backbone (5´ε → 3´δ). Pink steps show the modification position (left) and the overlay of all structure shows clear similarities (right). \u003cstrong\u003eb\u003c/strong\u003e. Backbone overlay comparing the LNA-amide-LNA step in ON30\u003csup\u003exLNA-Am-LNA\u003c/sup\u003e (purple) with the phosphodiester in ON26\u003csup\u003exDNA\u003c/sup\u003e (grey). \u003cstrong\u003ec\u003c/strong\u003e. Overlay of all amide backbones with or without LNA modifications (light blue = ON28\u003csup\u003exDNA-Am-DNA\u003c/sup\u003e, dark blue = ON29\u003csup\u003exLNA-Am-DNA\u003c/sup\u003e, purple = ON30\u003csup\u003exLNA-Am-LNA\u003c/sup\u003e).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/142ee56c5b07b98bf688bb82.png"},{"id":21082027,"identity":"6354b207-7083-4e9a-b82a-2822dbbdb200","added_by":"auto","created_at":"2022-05-04 19:08:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":150868,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eLNA-flanked amides and PS linkages increase gymnotic delivery, exon-skipping activity and reduce toxicity. In all cases luciferase activity was measured and normalised to both protein quantity and untreated cells. Experimental details and sequences are given in the Supplementary Information. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ea.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e HeLa pLuc/705 cells were transfected at 100 nM ON using LF2000. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eb.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Dose response for active ONs in \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ea\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ec.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Cells were treated with ONs without transfection agent. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ed.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Viability of the HeLa pLuc/705 cells following lipofection with ONs using LF2000 determined using WST-1 assay. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ee.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Change in cell viability after treatment with 200 nM ONs complexed with LF2000 compared to untreated, scale bar = 200 mm. For all graphs (\u003c/em\u003e\u003cstrong\u003e\u003cem\u003ea-d\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e)\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e \u003c/em\u003e\u003c/strong\u003e\u003cem\u003eData are means ± SD and values for ON18\u003c/em\u003e\u003csup\u003e\u003cem\u003e2¢OMe/4LAL/13PS \u003c/em\u003e\u003c/sup\u003e\u003cem\u003eand ON20\u003c/em\u003e\u003csup\u003e\u003cem\u003e2¢OMe/17PS \u003c/em\u003e\u003c/sup\u003e\u003cem\u003ewere compared by t test analysis. ns represents P \u0026gt; 0.05, * represents P \u0026lt; 0.05, ** represents P \u0026lt; 0.01, *** represents P \u0026lt; 0.001. Each dot represents one distinct replicate (n = 3). ON31\u003c/em\u003e\u003csup\u003e\u003cem\u003e2¢OMe/17PS\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u003csup\u003e\u003cem\u003escrambled\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e: CCUCAUUCACUCGAUUCA. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ef.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Reversed-phase HPLC chromatogram and \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eg.\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Electrospray (ES-) mass spectrum of purified ON18 used in the assays (required mass = 5976.2, found mass = 5975.5).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/8c4cfd6d0a741d64c4360e3c.png"},{"id":23797739,"identity":"44025607-f258-4e02-b98a-0a6786dfecc8","added_by":"auto","created_at":"2022-07-13 07:11:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1103759,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/9e4d93c9-5fe9-4c11-82c0-eb2e402f7ff4.pdf"},{"id":21082026,"identity":"07101bea-7c48-432f-a72f-c02b66ee8686","added_by":"auto","created_at":"2022-05-04 19:08:37","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20544,"visible":true,"origin":"","legend":"source data for Figure 6 and Supplementary Figure 25","description":"","filename":"Sourcedata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/dea5ac7797b63f2a8ce31b15.xlsx"},{"id":21081782,"identity":"2dca6381-3ca5-4048-9217-589518a4d4e6","added_by":"auto","created_at":"2022-05-04 18:58:37","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5544945,"visible":true,"origin":"","legend":"Supporting information","description":"","filename":"supportinginformationforNatCommunrevised.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/0c6533d0d765a78da3e5b88c.pdf"},{"id":21082006,"identity":"d50e0a21-e956-4fd2-af94-c4b886535cd8","added_by":"auto","created_at":"2022-05-04 19:03:37","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5346270,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"RS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/3ceae4bee06b06524c05aebf.pdf"},{"id":21081778,"identity":"ce3bc322-5924-424a-a329-96f745a12481","added_by":"auto","created_at":"2022-05-04 18:58:37","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":62109,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-1542733/v1/7eb8dd381b9b27f5db009d01.png"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nT.B.is a consultant to ATDBio Ltd.","formattedTitle":"Oligonucleotide analogues with locked-amide linkages have therapeutic potential","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntisense oligonucleotides (ASOs)\u0026nbsp;are synthetic nucleic acid analogues that bind to RNA to regulate gene expression and interfere with protein synthesis\u003csup\u003e1, 2\u003c/sup\u003e. Their modes of action include steric blocking of translation\u003csup\u003e3\u003c/sup\u003e, recruitment of RNase‑H for degradation of mRNA\u003csup\u003e4\u003c/sup\u003e, modulation of pre-mRNA splicing\u003csup\u003e5-7\u003c/sup\u003e and siRNA mediated gene silencing\u003csup\u003e8\u003c/sup\u003e. Therapeutic oligonucleotides in general have shown great promise as precision medicines, and have the potential to cure cancers, genetic disorders, and infectious diseases. They are particularly attractive because there are far more RNA targets than conventional protein targets in currently undruggable diseases\u003csup\u003e9\u003c/sup\u003e. Other advantages include their simple design criteria based on Watson-Crick base pairing and high target specificity.\u0026nbsp;However, although the ASO concept was first demonstrated as far back as 1978\u003csup\u003e10\u003c/sup\u003e, ASOs have only recently started to deliver on their initial promise. Fifteen oligonucleotides have been approved for clinical use (twelve since 2016) indicating their increasing therapeutic value.\u003csup\u003e11\u003c/sup\u003e Until recently all of these were for rare life limiting diseases, but the field just received a major boost with the EMA and NICE approval of the Novartis siRNA drug Inclisiran (Leqvio) for lowering LDL cholesterol levels in patients with hypercholesterolemia\u003csup\u003e12\u003c/sup\u003e.\u0026nbsp;Unlike previous oligonucleotide therapeutics, Inclisiran is used to treat a very common chronic disease. Although a major triumph, and an insight into what the future could hold for oligonucleotide therapeutics, Inclisiran is a special case where targeted delivery to the liver is required. Other organs are more difficult to penetrate, and inefficient biodistribution, poor cellular delivery, and toxicity prevent the wider adoption of antisense technology. Given these limitations, new oligonucleotide analogues are urgently needed to advance the field.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo achieve a therapeutic response an oligonucleotide must be stable \u003cem\u003ein vivo\u003c/em\u003e, bind to its target RNA with high selectivity and affinity, and display good pharmacokinetic properties\u003csup\u003e2\u003c/sup\u003e. Unmodified oligonucleotides are rapidly digested by nucleases in cells and are therefore completely unsuitable. Modifications designed to provide nuclease resistance\u003csup\u003e13\u003c/sup\u003e include 2\u0026acute;-OMe, 2\u0026acute;-\u003cem\u003eO\u003c/em\u003e-(2-methoxyethyl), 2\u0026acute;-fluoro sugars\u003csup\u003e14-16\u003c/sup\u003e, and phosphorothioate (PS) backbones (Fig. 1a)\u003csup\u003e15, 17\u003c/sup\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThe PS group improves cell uptake but reduces RNA target affinity, which can be restored by the 2\u0026acute;-sugar substituents\u003csup\u003e2\u003c/sup\u003e. Whilst these modifications have led to oligonucleotide therapies\u003csup\u003e11\u003c/sup\u003e, limited efficacy, off-target effects and toxicity remain obstacles to wider adoption in the clinic\u003csup\u003e18\u003c/sup\u003e. \u0026nbsp;Poor uptake into cells is particularly troublesome; less than 1% of an administered oligonucleotide typically reaches its target\u003csup\u003e19\u003c/sup\u003e.\u0026nbsp;Reducing the net anionic charge of oligonucleotides by partial or complete replacement of the phosphodiester backbone with neutral linkages would seem to be a viable strategy for increasing cell permeability and nuclease resistance\u003csup\u003e13, 20\u003c/sup\u003e. A clinically relevant example\u003csup\u003e21\u003c/sup\u003e is the phosphorodiamidate morpholino backbone (PMO) (Fig. 1a), but limitations in solid phase syntheses complicate the assembly of charge-reduced PMO\u0026ndash;DNA chimeras. Most other uncharged backbones that have been studied reduce the affinity and selectivity of therapeutic oligonucleotides towards their RNA targets\u003csup\u003e13\u003c/sup\u003e. However, we felt that the \u0026lsquo;reduced charge\u0026rsquo; principle warranted detailed investigation. Oligonucleotides containing the artificial amide backbone AM1 (Fig. 1b) are of interest as they form moderately stable duplexes with complementary RNA\u003csup\u003e22\u003c/sup\u003e \u003csup\u003e23-27\u003c/sup\u003e. Despite this, the required monomers have not been synthesised for all four canonical nucleobase analogues\u003csup\u003e28\u003c/sup\u003e. Inspired by the extremely high target affinity of locked nucleic acids (LNA) (Fig. 1b) developed in the 1990\u0026rsquo;s\u003csup\u003e29-32\u003c/sup\u003e, we hypothesised that conformationally locking the nucleosides surrounding the AM1 linkage would produce reduced-charge oligonucleotides (ONs) with higher target affinity, and in combination with PS linkages might yield ASOs with superior cell uptake and \u003cem\u003ein vivo\u003c/em\u003e stability. \u0026nbsp;Although attaching LNA sugars directly to other non-phosphorus-based charge-neutral backbones has so far failed to produce analogues with increased duplex stability\u003csup\u003e33-38\u003c/sup\u003e we reasoned that AM1, being a particularly close analogue of the phosphodiester backbone\u003csup\u003e39\u003c/sup\u003e, might behave differently (Fig. 1c).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere, we describe the synthesis of oligonucleotides containing LNA-amide linkages and show that these can be combined with phosphodiester or phosphorotioate backbones. We demonstrate the excellent duplex stabilisation of the LNA-amide linkages, mismatch discrimination and extreme resistance to nucleases. Preliminary studies show that LNA-amide ONs containing PS backbones have improved cellular uptake and are highly effective in exon-skipping assays.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLNA-amide monomer synthesis is efficient and scalable\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEfficient synthesis of chemically modified oligonucleotides is essential to fuel fundamental studies and therapeutic applications. We have devised a strategy which enables the straightforward assembly of oligonucleotides containing LNA-flanked amides which could be easily automated and scaled up. A maximum of eight monomers are required to make any sequence that contains non-contiguous amide linkages (four carboxylic acids and four amines).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAttaching the required ethanoic acid moiety to the 3\u0026acute;-carbon of the LNA sugar is challenging as it requires removal of the\u0026nbsp;3\u0026acute;-oxygen and formation of a C-C bond.\u0026nbsp;However, we were able to produce the required 5\u0026acute;-dimethoxytrityl (DMT)-protected 3\u0026acute;-ethanoic acid\u0026nbsp;LNA-monomers in 8 steps from \u003cstrong\u003e1\u003c/strong\u003e, the same number as for conventional LNA phosphoramidites\u003csup\u003e43\u003c/sup\u003e (Fig. 2a). Moreover, we did this with minimal chromatography. We first built the sugar with the C3\u0026acute;-ester \u003cstrong\u003e5\u003c/strong\u003e, before addition of the nucleobase. This approach avoids complications associated with forming a C-C bond at the 3\u0026acute;-side of a nucleoside using the Barton-McCombie reaction\u003csup\u003e23-25, 44\u003c/sup\u003e or the hydrogenation step if a Wittig reaction is used\u003csup\u003e45, 46\u003c/sup\u003e, which would ultimately limit the range of heterocyclic bases that can be added. Key intermediate \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003ewas readily prepared on multiple gram scale in 81% overall yield from commercially available \u003cstrong\u003e1\u0026nbsp;\u003c/strong\u003ewithout the need for chromatographic separation as follows. Hydrogenolysis of compound \u003cstrong\u003e1\u003c/strong\u003e afforded alcohol \u003cstrong\u003e2\u0026nbsp;\u003c/strong\u003ewhich was subsequently oxidised\u0026nbsp;to give compound \u003cstrong\u003e3\u003c/strong\u003e. Olefination of \u003cstrong\u003e3\u0026nbsp;\u003c/strong\u003ewith\u0026nbsp;(carbethoxymethylene)triphenylphosphorane (Wittig reaction) selectively yielded \u003cstrong\u003e4\u0026nbsp;\u003c/strong\u003eas the (\u003cem\u003eE\u003c/em\u003e)-stereoisomer (Fig. 2b). Catalytic hydrogenation of \u003cstrong\u003e4\u003c/strong\u003e using Pd/C and H\u003csub\u003e2\u003c/sub\u003e gave \u003cstrong\u003e5\u003c/strong\u003e as a single stereoisomer (Fig. 2b). This stereoselectivity was predicted because the\u0026nbsp;1,2-\u003cem\u003eO\u003c/em\u003e-isopropylidene groups on the \u0026alpha;-face of furanosyl carbohydrate derivatives direct incoming H\u003csub\u003e2\u003c/sub\u003e to the \u0026beta;-face\u003csup\u003e47\u003c/sup\u003e. Key intermediate \u003cstrong\u003e5\u0026nbsp;\u003c/strong\u003ewas then converted to the 1,2-di-\u003cem\u003eO\u003c/em\u003e-acetate glycosyl donor \u003cstrong\u003e6\u0026nbsp;\u003c/strong\u003efollowing a\u0026nbsp;procedure reported by Arzel \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e48\u003c/sup\u003e, avoiding the formation of a lactone which occurred when the acetonide was cleaved in the presence of water.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pathways to each monomer then diverged, with Vorbr\u0026uuml;ggen conditions\u003csup\u003e43, 49\u003c/sup\u003e utilised for addition of the nucleobases to access \u003cstrong\u003e7a-e\u003c/strong\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eSubsequent simultaneous unmasking of the 3\u0026acute;-carboxyl and 2\u0026acute;-hydroxyl groups by treatment with hydroxide, followed by cyclisation to form the 2\u0026acute;- 4\u0026acute;-oxymethylene bridge, then 5\u0026acute;-mesyl deprotection,\u0026nbsp;gave the hydroxy-LNA acid compounds \u003cstrong\u003e8a-e\u003c/strong\u003e. The progress of the reaction was rapid; mesyl deprotection using hydroxide ion conventionally requires several days under reflux conditions\u003csup\u003e43\u003c/sup\u003e. We postulate that the acceleration in rate is due to neighbouring group participation whereby the carboxylate anion displaces the 5\u0026acute;-mesyl\u0026nbsp;group, forming a lactone that is subsequently opened by hydrolysis (Supplementary Fig. 1). Finally, we treated the resulting hydroxy-acids \u003cstrong\u003e8a-e\u003c/strong\u003e with 4,4\u0026acute;-dimethoxytrityl chloride (DMT-Cl) in pyridine to give the DMT-protected LNA analogue nucleosides \u003cstrong\u003e9a-e\u003c/strong\u003e\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eUsing this strategy we were able to access all four canonical nucleoside analogues along with the 5-methylcytidine monomer which is used in place of cytidine in antisense experiments to increase target affinity and improve other therapeutic properties\u003csup\u003e18\u003c/sup\u003e. Additionally, we required 5\u0026acute;-MMT-amino LNA phosphoramidite \u003cstrong\u003e10\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWhilst this had been previously synthesised\u003csup\u003e40\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewe chose to develop a more efficient route (Supplementary Fig. 2). Commercially available 5\u0026acute;-MMT-amino dT\u003cstrong\u003e\u0026nbsp;11\u003c/strong\u003e and 5\u0026acute;-DMT thymidine-3\u0026acute;‑ethanoic acid \u003cstrong\u003e12\u003c/strong\u003e\u003csup\u003e41, 42\u003c/sup\u003e (Fig. 2c) were also required to enable us to make oligonucleotides to compare the properties of DNA amide with those of LNA-amide.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eChimeric oligonucleotides can be synthesised in high purity\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur oligonucleotide synthesis strategy is shown in Fig. 3. A phosphoramidite monomer with an MMT-protected 5\u0026acute;-amino group, either LNA \u003cstrong\u003e10\u003c/strong\u003e\u003csup\u003e40\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eor deoxythymidyl \u003cstrong\u003e11\u003c/strong\u003e, is added to the oligonucleotide, and the amine is deprotected using trichloroethanoic acid. An LNA-acid (or DNA-acid\u003csup\u003e50\u003c/sup\u003e) monomer is coupled to the free amine using PyBOP activating agent in the presence of a non-nucleophilic base (\u003cem\u003eN\u003c/em\u003e-methylmorpholine) to form the amide bond. Oligonucleotide synthesis is then resumed, starting with removal of the DMT group.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe process is repeated to install multiple non-contiguous amides in the same oligonucleotide. To demonstrate this, DMT-protected LNA acids \u003cstrong\u003e9a-e\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ephosphoramidites \u003cstrong\u003e10\u003c/strong\u003e and \u003cstrong\u003e11\u003c/strong\u003e, and DNA acid \u003cstrong\u003e12\u003c/strong\u003e\u003csup\u003e41, 42\u003c/sup\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Fig. 2), were used to synthesise several oligonucleotides, some of which contain multiple additions of LNA-amide, 2\u0026acute;-OMe sugars and PS linkages (Supplementary Table 1). In all cases, we obtained the oligonucleotides in high purity. High-performance liquid chromatography (HPLC) and mass spectrometry demonstrated the high incorporation efficiency for the DMT-protected LNA acids \u003cstrong\u003e9a-e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Supplementary Fig. 3-7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLNA sugars stabilise duplexes containing the amide backbone\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the ability of the LNA-amide combination to bind to complementary RNA with high affinity we synthesised a series of 13-mer ONs with a central amide with a T-T sequence. These constructs were composed of either no LNA (ON1\u003csup\u003eDNA-Am-DNA\u003c/sup\u003e), an LNA 5\u0026acute; to the amide (ON2\u003csup\u003eLNA-Am-DNA\u003c/sup\u003e), an LNA 3\u0026acute; to the amide (ON3\u003csup\u003eDNA-Am-LNA\u003c/sup\u003e), or LNA on both sides of the amide (ON4\u003csup\u003eLNA-Am-LNA\u003c/sup\u003e, Fig. 4a). Controls without amide (ON5\u003csup\u003eLNA-LNA\u003c/sup\u003e and ON6\u003csup\u003eDNAcontrol\u003c/sup\u003e) were also made. We compared duplex denaturation temperatures (\u003cem\u003eT\u003c/em\u003e\u003csub\u003em\u003c/sub\u003es) after hybridisation with DNA and RNA complementary strands (Fig. 4b, Supplementary Table 2, Supplementary Fig. 8 and 9). ON2\u003csup\u003eLNA‑Am-DNA\u003c/sup\u003e produced a significant increase in DNA:RNA hybrid stability, (+3.0 ˚C) compared to the unmodified ON6\u003csup\u003eDNAcontrol\u003c/sup\u003e, and an increase of +3.4˚C compared to \u0026lsquo;amide only\u0026rsquo; ON1\u003csup\u003eDNA-Am-DNA\u003c/sup\u003e. Importantly, ON4\u003csup\u003eLNA‑Am-LNA\u003c/sup\u003e, in which the amide is surrounded by LNA sugars, gave the greatest increase in stability (+5.1 ˚C). It is noteworthy that ON2\u003csup\u003eLNA-Am-DNA\u003c/sup\u003e and ON4\u003csup\u003eLNA-Am-LNA\u003c/sup\u003e provide the first examples of duplex stabilisation by an LNA sugar attached to a non-phosphorus artificial DNA backbone. The RNA target selectivity of LNA-amide-containing ONs was excellent; a single mismatched base pair greatly reduced duplex stability, in some cases by \u0026gt;14 ˚C (Supplementary Table 2, Supplementary Fig. 10-13) In summary, an amide linkage flanked by LNA on both sides gives strong DNA:RNA duplex stabilisation and good mismatch discrimination.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn duplexes with DNA targets, ONs with all combinations of LNA and DNA sugars around the amide linkage were slightly destabilising (between ‑0.1 ˚C to ‑2.6 ˚C), indicating the selectivity of the amide linkage for complementary RNA. The stabilisation induced by the LNA amide combination is cumulative and general (Fig. 4c, Supplementary Table 3, Supplementary Fig. 14-17). In a biologically relevant sequence context, four LNA-amides increase duplex stability against complementary RNA by an impressive 13.0 ˚C compared to only 5.1 ˚C for the DNA target. \u0026nbsp;This large difference is important when developing oligonucleotides to interact with RNA. Oligonucleotides for \u003cem\u003ein vivo\u003c/em\u003e studies usually contain 2\u0026acute;-OMe modified sugars and/or phosphorothioate backbones to prevent degradation by nucleases. \u0026nbsp;In such oligonucleotides the combination of LNA and amide also greatly increases duplex\u0026nbsp;stability (Supplementary Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCombining LNA and amide provides strong nuclease resistance\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTherapeutic oligonucleotides must remain stable in cells for prolonged periods to remain active. To evaluate whether the combination of LNA and amide confers greater nuclease resistance than LNA alone, we incubated unmodified DNA (ON15\u003csup\u003eDNA/17PO\u003c/sup\u003e) and DNA with four LNA-amide linkages (ON14\u003csup\u003e\u0026nbsp;DNA/4LAL/13PO\u003c/sup\u003e) in a 1:1 mixture of phosphate buffered saline (PBS) and foetal\u0026nbsp;bovine serum (FBS) to mimic the \u003cem\u003ein vivo\u003c/em\u003e environment, and\u0026nbsp;compared it to the equivalent construct with LNA but without amide linkages (ON25\u003csup\u003eDNA/8LNA/17PO\u003c/sup\u003e,\u0026nbsp;Supplementary Fig. 18). The results show that the combination of LNA and amide confers extreme resistance to nucleases. Both the oligonucleotides lacking amide linkages had partially degraded within 1 hour whereas ON14\u003csup\u003eDNA/4LAL/13PO\u003c/sup\u003e remained intact after 8 hours. Interestingly ON14 shows stability at its 3\u0026acute;-end, even though this region has an unmodified sugar phosphate trimer. The 3\u0026acute;-terminal pentamer region, however, is highly modified. It has reduced charge due to the amide linkage and also contains two LNA sugars. It may therefore not be recognised by nucleases. This enhanced stability further illustrates the advantages of removing charge and including modified sugars in antisense oligonucleotides.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eX-ray crystallography of the LNA-amide modification\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe solved several X-ray structures to determine the effects of LNA and amide modifications on duplex structure and conformation. These are the first crystal structures of DNA:RNA hybrids that contain amide linkages. An amide-modified RNA:RNA duplex was analysed previously, but this had amides in \u003cem\u003eboth\u003c/em\u003e strands surrounded by multiple mismatched base pairs which cannot exist outside the solid-state at ambient temperature\u003csup\u003e27\u003c/sup\u003e. The sequence of the modified DNA:RNA hybrid duplexes (Supplementary Table 4) was based on the corresponding unmodified version (d-CT\u003cu\u003eTT\u003c/u\u003eTCTTTG/rCAAAGAAAAG)\u003csup\u003e51\u003c/sup\u003e (the location of the amide is underlined). Good quality crystals diffracting between 2.5-2.8 \u0026Aring; resolution were obtained for the DNA:RNA hybrid in which the DNA strand contains an amide linkage flanked by DNA on both sides, LNA on both sides and with LNA only on the 5\u0026acute;-side (Supplementary Fig. 19). The unmodified DNA:RNA duplex was also studied. The data collection and refinement statistics are given in Supplementary Table 5. Electron density maps at the modification position for the four nucleic acid crystal structures reported in this manuscript are given in Supplementary Fig. 20.\u003c/p\u003e\n\u003cp\u003eThe hybrids with amide and LNA-amide backbones (Fig. 5a) are structurally very similar to the unmodified duplex (all-atom RMSD 0.4 \u0026Aring;) as shown by their superimposition (Fig. 5a, Supplementary Fig. 21). All structures adopt the A-conformation with sugar puckers clustering around C3\u0026acute;-\u003cem\u003eendo\u003c/em\u003e (Supplementary Fig. 22). As expected, all duplexes are stabilised by canonical Watson-Crick base pairs, indicating that the thermodynamic improvements due to the LNA-amide backbones are not due to unusual changes in hydrogen bonding interactions. In agreement with the DNA:RNA hybrid NMR structure by Rosners\u003csup\u003e22\u003c/sup\u003e in which the DNA strand contained multiple amides, our X-ray studies indicate that the amide linkage is a close mimic of the phosphodiester backbone (Fig. 5b). Both are four-atom linkages, hence similar in length, and the amide carbonyl is orientated in the same direction as one of the phosphodiester P‑O bonds.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn Fig. 5c, the structures of all amide backbones are overlaid to assess the effects of the LNA modifications. Between each structure, the orientation of the backbone is consistent, directing the amide oxygen into the major groove. Other atomic positions of the backbones also show close similarity, and the presence of 3\u0026acute;-LNA causes no significant distortion. 5\u0026acute;-LNA does however cause some structural displacement; the 5\u0026acute;-sugars in the LNA-amide-DNA and LNA-amide-LNA structures are shifted slightly outwards compared to the DNA-amide-DNA and unmodified strands. Despite this, the positioning of the amide backbone remains consistent between each structure. The amide adopts the expected \u003cem\u003etrans-\u003c/em\u003econformation, and LNA on the 5\u0026acute;-side of the amide has little effect on backbone torsion angles (Fig. 5c, Supplementary Fig. 23). In summary, combined LNA and amide modifications have minimal effect on the duplex structure, and are excellent mimics of natural phosphodiesters.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCombining LNA-amide and PS enhances gymnotic delivery\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a preliminary study we have evaluated the biological activity of the LNA-amide combination using the HeLa pLuc/705 cell line\u003csup\u003e52\u003c/sup\u003e that carries a luciferase-encoding gene interrupted by a mutated \u0026szlig;-globin intron\u003csup\u003e52\u003c/sup\u003e. This mutation creates a 5\u0026acute;-splice site which activates a cryptic 3\u0026acute;-splice site, resulting in incorrect mRNA splicing and the production of non-functional luciferase. An oligonucleotide that hybridises to the mutant\u0026nbsp;5\u0026acute;-splice site prevents incorporation of the aberrant intron, restoring the pre-mRNA splicing to produce functional luciferase, which is quantified by luminometry. Oligonucleotides complementary to this aberrant splice site were synthesised with combinations of different modifications\u0026nbsp;to determine their individual effects (Supplementary Table 3).\u0026nbsp;ON14\u003csup\u003eDNA/4LAL/13PO\u003c/sup\u003e,\u0026nbsp;ON16\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/4LAL/13PO\u003c/sup\u003e, and\u0026nbsp;ON18\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/4LAL/13PS\u003c/sup\u003e were designed to evaluate LNA-amide with the DNA, 2\u0026acute;‑OMe/phosphodiester, and 2\u0026acute;-OMe/phosphorothioate backbones respectively, and ONs 14 and 16 were included to determine the degree to which LNA-amide influences\u0026nbsp;delivery, activity and toxicity\u0026nbsp;in the absence of PS linkages. LNA and amide linkages are incompatible with RNase-H so there is no risk of ON14 or ON16 inadvertently destroying the RNA target\u003csup\u003e2,56\u003c/sup\u003e. Three controls were included:\u0026nbsp;ON20\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/17PS\u003c/sup\u003e (which represents the gold standard in the assay) to determine whether LNA-amide enhances biological activity\u003csup\u003e52\u003c/sup\u003e,\u0026nbsp;ON17\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/17PO\u0026nbsp;\u003c/sup\u003e to evaluate the effects of the PS linkage independently of LNA or amide linkages, and\u0026nbsp;ON19\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/8LNA/17PS\u003c/sup\u003e with LNA sugars but no amide linkages to determine the effects of the enhanced duplex stability caused by LNA. \u0026nbsp;A scrambled control with a 2\u0026acute;-OMe/PS backbone was also included to determine off target effects (ON31\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/17PS\u003c/sup\u003e \u003csup\u003escrambled\u003c/sup\u003e, Fig. 6).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo compare biological activity independent of cell uptake, Lipofectamine 2000 (LF2000), a cationic liposome transfection/delivery reagent, was used. All three target-complementary PS-ONs were active in the assay (ON20\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/17PS\u003c/sup\u003e,\u0026nbsp;ON18\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/4LAL/13PS\u003c/sup\u003e, and\u0026nbsp;ON19\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/8LNA/17PS\u003c/sup\u003e), whereas all\u0026nbsp;the\u0026nbsp;PO-ONs (ON14\u003csup\u003eDNA/4LAL/13PO\u003c/sup\u003e,\u0026nbsp;ON16\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/4LAL/13PO\u003c/sup\u003e and\u0026nbsp;ON17\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/17PO\u003c/sup\u003e)\u0026nbsp;were inactive at 100 nM (Fig. 6a). Hence, in agreement with previous studies, the phosphorothioate modification in a target-complementary sequence is necessary for splice-switching activity. This could result from the PS groups enhancing nuclear enrichment\u003csup\u003e53\u003c/sup\u003e of the oligonucleotides, and/or recruiting ILF2/3 to the RNA transcript\u003csup\u003e54\u003c/sup\u003e. Notably, the addition of the amide linkage significantly improved the splice-switching activity of 2\u0026acute;-OMe/PS ONs at the lower concentrations (6.25 nM and 12.5 nM), probably due to improved target affinity (Fig. 6b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we compared the naked (gymnotic) uptake of the ONs. These conditions more closely represent \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eapplications where transfection agents such as LF2000 cannot be used. We seeded cells at low confluency, added the oligonucleotides in fresh media after 16 h, and measured luciferase activity after a further 96 h. The presence of just four LNA-amides (ON18\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/4LAL/13PS\u003c/sup\u003e) significantly increased activity in a dose-dependent manner compared to ON20\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/17PS\u003c/sup\u003e (Fig. 6c). Greater than 5-fold increase in activity was observed for gymnotic delivery compared to a maximum of 3-fold increase for the LF2000‑mediated transfection. This suggests that synergy between the PS and LNA-amide modifications leads to enhanced productive delivery into cells. The improved therapeutic properties of LNA-amide/PS oligonucleotides observed in these preliminary studies are possibly due to a combination of reduced charge from the LNA-amides and interactions of the PS backbone with cellular components. Improved cell uptake may result from the neutral amide linkages breaking up the poly-anionic backbone into short segments which penetrate cells more readily than long poly-anionic stretches. Interestingly, ON19\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/8LNA/PS\u0026nbsp;\u003c/sup\u003ewith LNA and no amides showed only slight dose response in activity, even at the highest concentration tested (Fig. 6c, Supplementary Fig. 24). This could be due to its binding to off-targets, altered rigidity, or undesirable secondary structures induced by the extreme stability caused by the LNA sugars, reducing the ability of the ON to interact with the cell surface, a mechanism for productive uptake.\u0026nbsp;ON16\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/4LAL/13PO\u003c/sup\u003e with LNA-amides and no PS linkages also displayed slight gymnotic splice-switching activity (Supplementary Fig. 24).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe compared the viability of the HeLa cells following lipofection using a WST-1 cell proliferation assay (Fig. 6d). At the highest concentration tested (400 nM) the cells treated with ON20\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/17PS\u003c/sup\u003e were only 21% viable, whereas the cells treated with the same concentration of ON18\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/4LAL/13PS\u003c/sup\u003e were 50% viable, demonstrating that the LNA-amide linkage significantly reduces the cytotoxicity of ONs delivered with LF2000. This is verified by analysis of the protein levels (Supplementary Fig. 25) and visible cell death (Fig. 6e, and Supplementary Fig. 26). It supports the use of the combination of LNA-amide, 2\u0026acute;-OMe and PS modifications for \u003cem\u003ein-vitro\u003c/em\u003e studies. Interestingly, the oligonucleotide containing eight LNA sugars without amide linkages (ON19\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/8LNA/17PS\u003c/sup\u003e) had a poor toxicity profile. This could be due to off-target effects and could also explain why, despite showing the highest affinity towards RNA in the UV melting studies, the LNA modified ON19\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e\u0026cent;\u003c/sup\u003e\u003csup\u003eOMe/8LNA/17PS\u003c/sup\u003e was not the most active in the exon-skipping assay. Further detailed studies are required to determine whether this is a general or a sequence specific phenomenon, and to validate its relevance in terms of toxicity.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven that cell uptake and toxicity remain major challenges when developing new therapeutic oligonucleotides the results in Fig. 6 suggest that our modification strategy could be advantageous. The synergistic effect of LNA-amide linkages and phosphorothioate modifications appear to produce oligonucleotides with enhanced biological properties. However, cell culture studies cannot address some of the major challenges in oligonucleotide therapeutics, particularly those that relate to pharmacokinetics, pharmacodynamics, biodistribution and aspects of toxicity. We are therefore planning further detailed biological studies on LNA-amide-phosphorothioate oligonucleotides to answer these important questions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe have 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 development. These new constructs have high resistance to enzymatic degradation and bind to complementary RNA with affinity and selectivity superior to unmodified ONs. The artificial backbone causes minimal structural deviation in DNA:RNA hybrids, consistent with their strong affinity for RNA. Oligonucleotides with alternating LNA-amide and phosphodiester (or phosphorothioate) backbones cannot give rise to recyclable LNA mononucleotides (modified dNTPs) in the presence of cellular nucleases, and their favourable toxicity profile relative to LNA in these initial studies may reflect this. In studies with gymnotic (naked) delivery, combining LNA-amides with phosphorothioates improves cell uptake. Poor cellular uptake is currently a major barrier in oligonucleotide therapeutics and combining the PS and LNA modifications with charge-neutral amide backbones such as AM1 could lead to improved clinical efficacy. Research is in progress to explore these new analogues in a range of cellular assays and in other therapeutic interventions such as siRNA and RNase-H mediated antisense inhibition.\u003c/p\u003e \u003cp\u003eFinally, artificial nucleic backbones are of interest to researchers in many other fields including nucleic acid chemistry, chemical biology, biochemistry, medicinal chemistry, diagnostics, gene synthesis, gene editing, nanotechnology, materials chemistry and biophysics. We hope that this work will catalyse future research in these areas.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAmide-modified oligonucleotide synthesis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eOligonucleotide segment synthesis.\u003c/em\u003e Oligonucleotide synthesis was performed on an Applied Biosystems 394 automated DNA/RNA synthesiser on a 1.0 \u0026micro;mole scale using a standard phosphoramidite cycle of detritylation, coupling, and oxidation. No capping step was used. All β-cyanoethyl phosphoramidite monomers were dissolved in anhydrous MeCN (10% CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e was added when 2\u0026prime;-OMe U phosphoramidite was used) to a concentration of 0.1 M immediately prior to use. 5-(Benzylthio)-1\u003cem\u003eH\u003c/em\u003e-tetrazole (BTT) activator (0.3 M) was used with a coupling time of 50 s for normal dA, dG, dC and T phosphoramidites, this was extended to 6 min for 2\u0026prime;-OMe and LNA phosphoramidites. Standard iodine oxidiser was used for phosphodiester oligonucleotides. For the phosphorothioate oligonucleotides, 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) was used as the sulfurisation agent, and the solid support was washed with MeCN after each phosphoramidite coupling before the sulfurisation step. Sulfurisation time was initially 3 min, and after this period fresh EDITH was sent to the synthesis column and left for another 3 min. Further details are given in the Supplementary Information.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmino monomer addition.\u003c/em\u003e The MMT-protected 5\u0026acute;-amino phosphoramidite monomer (either LNA \u003cb\u003e10\u003c/b\u003e\u003csup\u003e40\u003c/sup\u003e or commercially available deoxythymidyl \u003cb\u003e11\u003c/b\u003e) was dissolved in anhydrous MeCN to a concentration of 0.1 M immediately prior to use. The same coupling conditions as above were used, but the coupling time was extended to 10 min. The MMT protecting group was cleaved on the Applied Biosystems 394 automated synthesiser using 3% TCA in CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e with an extended cleavage time of 2 min. The solid support was then washed with acetonitrile on the synthesiser for 3 min. To improve the coupling efficiency in the next step, the solid support was washed with 0.5% (v/v) \u003cem\u003eN\u003c/em\u003e-methylmorpholine in DMF (1 x 1mL) followed by DMF (3 x 1 mL).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAmide bond formation on resin (peptide coupling).\u003c/em\u003e All amide couplings were performed manually on the synthesis column. A solution with 10 equivalents of acid monomer, 10 equivalents of PyBOP and 30 equivalents of \u003cem\u003eN\u003c/em\u003e-methylmorpholine was first prepared in 400 \u0026micro;L of DMF. This was then taken up into a 1 mL syringe and loaded onto the column before a second 1 mL syringe was attached to the other end of the synthesis column. The mixture was agitated every 10 min for 1 h. The columns were then washed with DMF (3 x 1 mL) followed by MeCN (5 x 1 mL) and dried by passing argon through the column. The column was then returned to the synthesiser to continue oligonucleotide synthesis.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCleavage of oligonucleotides from resin, deprotection and purification.\u003c/em\u003e LNA-amide containing oligonucleotides were isolated with the final 5\u0026prime;-DMT protecting group still in place (DMT-ON). Following solid phase synthesis, the cyanoethyl groups were removed by a 15 min treatment with 20% diethylamine in MeCN. The resin was then washed with MeCN (5 x 1 mL) and dried by passing a stream of argon through the synthesis column. The oligonucleotides were cleaved from the solid support and deprotected by heating in concentrated aqueous ammonia in a sealed glass vial at 55 \u0026deg;C for 5 hours. The ammonia was removed under reduced pressure prior to oligonucleotide purification. The DMT-ON oligonucleotides were purified by reverse-phase high performance liquid chromatography (RP-HPLC) and lyophilised. They were then dissolved in 0.5 mL of 80% acetic acid and left for 1 hour at room temperature to remove the DMT group. The solution was neutralised with 0.5 mL of triethylammonium acetate buffer (2 M, pH 7) and the detritylated oligonucleotides were desalted using a NAP-10 column (Cytiva) then freeze dried.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eCrystallographic data for the structures in this Article have been deposited at the Cambridge Crystallographic Data Centre under deposition numbers CCDC 2105684 and 2105685.DNA structural data obtained by X-ray crystallography were deposited in the Protein Data Bank (PDB) and are available with the following accession codes 7NRP, 7OOS, 7OZZ and 7OOO. Source data is provided for Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Supplementary Figs.\u0026nbsp;24 and 25. The authors declare that all other data supporting the findings of this study are available within the paper and its supplementary information files.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr Samir El-Andaloussi for the\u0026nbsp;HeLa pLuc/705 cell line\u003csup\u003e52\u003c/sup\u003e. This work was funded in part by a BBSRC research grant to TB (New oligonucleotide analogues for therapeutic applications. BB/S018794/1).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.R.B, L.M.P, L.L and P.K performed the small molecule synthesis; Y.R.B, L.M.P and K.E.C carried out the small molecule crystallography work; Y.R.B, A.E.S, J.C, S.E and D.S synthesised and purified the ONs; Y.R.B, J.C and L.C performed UV melting and enzymatic stability studies; C.T, M.A.McD, J.S.H and J.P.H carried out the ON XRD studies; Y.R.B, W.F.L and G.McC performed the cell studies; Y.R.B, A.E.S, P.K and T.B. designed the study. All authors contributed to the writing of the manuscript. T.B. oversaw and managed the project. M.J.A.W oversaw the biological work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.B.is a consultant to ATDBio Ltd.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSmith, C.I.E. \u0026amp; Zain, R. Therapeutic Oligonucleotides: State of the Art. Annu. Rev. Pharmacol. Toxicol. \u003cb\u003e59\u003c/b\u003e, 605\u0026ndash;630 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhvorova, A. \u0026amp; Watts, J.K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol. \u003cb\u003e35\u003c/b\u003e, 238\u0026ndash;248 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRinaldi, C. \u0026amp; Wood, M.J.A. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nature Reviews Neurology \u003cb\u003e14\u003c/b\u003e, 9\u0026ndash;21 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang, X.-H., Sun, H., Nichols, J.G. \u0026amp; Crooke, S.T. RNase H1-Dependent Antisense Oligonucleotides Are Robustly Active in Directing RNA Cleavage in Both the Cytoplasm and the Nucleus. Mol. Ther. \u003cb\u003e25\u003c/b\u003e, 2075\u0026ndash;2092 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDominski, Z. \u0026amp; Kole, R. Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cb\u003e90\u003c/b\u003e, 8673\u0026ndash;8677 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSierakowska, H., Sambade, M.J., Agrawal, S. \u0026amp; Kole, R. Repair of thalassemic human beta-globin mRNA in mammalian cells by antisense oligonucleotides. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cb\u003e93\u003c/b\u003e, 12840\u0026ndash;12844 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAartsma-Rus, A. et al. Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum. Mol. Genet. \u003cb\u003e12\u003c/b\u003e, 907\u0026ndash;914 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSetten, R.L., Rossi, J.J. \u0026amp; Han, S.-p. The current state and future directions of RNAi-based therapeutics. Nature Reviews Drug Discovery \u003cb\u003e18\u003c/b\u003e, 421\u0026ndash;446 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWarner, K.D., Hajdin, C.E. \u0026amp; Weeks, K.M. Principles for targeting RNA with drug-like small molecules. Nature Reviews Drug Discovery \u003cb\u003e17\u003c/b\u003e, 547\u0026ndash;558 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZamecnik, P.C. \u0026amp; Stephenson, M.L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cb\u003e75\u003c/b\u003e, 280\u0026ndash;284 (1978).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong, H., Veedu, R.N. \u0026amp; Diermeier, S.D. Recent Advances in Oligonucleotide Therapeutics in Oncology. Int. J. Mol. Sci. \u003cb\u003e22\u003c/b\u003e, 3295 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFitzgerald, K. et al. A Highly Durable RNAi Therapeutic Inhibitor of PCSK9. N. Engl. J. Med. \u003cb\u003e376\u003c/b\u003e, 41\u0026ndash;51 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreier, S.M. \u0026amp; Altmann, K.-H. The ups and downs of nucleic acid duplex stability: Structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. \u003cb\u003e25\u003c/b\u003e, 4429\u0026ndash;4443 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCummins, L.L. et al. Characterization of fully 2'-modified oligoribonucleotide hetero- and homoduplex hybridization and nuclease sensitivity. Nucleic Acids Res. \u003cb\u003e23\u003c/b\u003e, 2019\u0026ndash;2024 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonia, B.P., Johnston, J.F., Sasmor, H. \u0026amp; Cummins, L.L. Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras. J. Biol. Chem. \u003cb\u003e271\u003c/b\u003e, 14533\u0026ndash;14540 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin, P. A New Access to 2'-O-Alkylated Ribonucleosides and Properties of 2'-O-Alkylated Oligoribonucleotides. Helv. Chim. Acta \u003cb\u003e78\u003c/b\u003e, 486\u0026ndash;504 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. \u003cb\u003e24\u003c/b\u003e, 374\u0026ndash;387 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts, T.C., Langer, R. \u0026amp; Wood, M.J.A. Advances in oligonucleotide drug delivery. Nature Reviews Drug Discovery \u003cb\u003e19\u003c/b\u003e, 673\u0026ndash;694 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGodfrey, C. et al. Delivery is key: lessons learnt from developing splice-switching antisense therapies. EMBO Mol. Med. \u003cb\u003e9\u003c/b\u003e, 545\u0026ndash;557 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeade, B.R. et al. Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications. Nat. Biotechnol. \u003cb\u003e32\u003c/b\u003e, 1256\u0026ndash;1261 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAartsma-Rus, A. \u0026amp; Krieg, A.M. FDA Approves Eteplirsen for Duchenne Muscular Dystrophy: The Next Chapter in the Eteplirsen Saga. Nucleic Acid Ther. \u003cb\u003e27\u003c/b\u003e, 1\u0026ndash;3 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelvam, C., Thomas, S., Abbott, J., Kennedy, S.D. \u0026amp; Rozners, E. Amides as Excellent Mimics of Phosphate Linkages in RNA. Angew. Chem. Int. Ed. \u003cb\u003e50\u003c/b\u003e, 2068\u0026ndash;2070 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIdziak, I., Just, G., Damha, M.J. \u0026amp; Giannaris, P.A. Synthesis and hybridization properties of amide-linked thymidine dimers incorporated into oligodeoxynucleotides. Tetrahedron Lett. \u003cb\u003e34\u003c/b\u003e, 5417\u0026ndash;5420 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebreton, J., Waldner, A., Lesueur, C. \u0026amp; De Mesmaeker, A. Antisense Oligonucleotides with Alternating Phosphodiester-\"Amide-3\" Linkages. \u003cem\u003eSynlett\u003c/em\u003e 1994, 137\u0026ndash;140 (1994).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Mesmaeker, A. et al. Amides as a New Type of Backbone Modification in Oligonucleotides. Angew. Chem. Int. Ed. \u003cb\u003e33\u003c/b\u003e, 226\u0026ndash;229 (1994).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHardcastle, T. et al. A Single Amide Linkage in the Passenger Strand Suppresses Its Activity and Enhances Guide Strand Targeting of siRNAs. ACS Chem. Biol. \u003cb\u003e13\u003c/b\u003e, 533\u0026ndash;536 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMutisya, D. et al. Amide linkages mimic phosphates in RNA interactions with proteins and are well tolerated in the guide strand of short interfering RNAs. Nucleic Acids Res. \u003cb\u003e45\u003c/b\u003e, 8142\u0026ndash;8155 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKotikam, V. \u0026amp; Rozners, E. Amide-Modified RNA: Using Protein Backbone to Modulate Function of Short Interfering RNAs. Acc. Chem. Res. \u003cb\u003e53\u003c/b\u003e, 1782\u0026ndash;1790 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHagedorn, P.H. et al. Locked nucleic acid: modality, diversity, and drug discovery. Drug Discovery Today \u003cb\u003e23\u003c/b\u003e, 101\u0026ndash;114 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh, S.K., A. Koshkin, A., Wengel, J. \u0026amp; Nielsen, P. LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem. Commun., 455\u0026ndash;456 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh, S.K. \u0026amp; Wengel, J. Universality of LNA-mediated high-affinity nucleic acid recognition. Chem. Commun., 1247\u0026ndash;1248 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBondensgaard, K. et al. Structural Studies of LNA:RNA Duplexes by NMR: Conformations and Implications for RNase H Activity. Chem. Eur. J. \u003cb\u003e6\u003c/b\u003e, 2687\u0026ndash;2695 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma, V.K. et al. Synthesis and biological properties of triazole-linked locked nucleic acid. Chem. Commun. \u003cb\u003e53\u003c/b\u003e, 8906\u0026ndash;8909 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, P., Truong, L., Baker, Y.R., El-Sagheer, A.H. \u0026amp; Brown, T. Synthesis, Affinity for Complementary RNA and DNA, and Enzymatic Stability of Triazole-Linked Locked Nucleic Acids (t-LNAs). ACS Omega \u003cb\u003e3\u003c/b\u003e, 6976\u0026ndash;6987 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, P., El-Sagheer, A.H., Truong, L. \u0026amp; Brown, T. Locked nucleic acid (LNA) enhances binding affinity of triazole-linked DNA towards RNA. Chem. Commun. \u003cb\u003e53\u003c/b\u003e, 8910\u0026ndash;8913 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThorpe, C., Epple, S., Woods, B., El-Sagheer, A.H. \u0026amp; Brown, T. Synthesis and biophysical properties of carbamate-locked nucleic acid (LNA) oligonucleotides with potential antisense applications. Org. Biomol. Chem. \u003cb\u003e17\u003c/b\u003e, 5341\u0026ndash;5348 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLauritsen, A. \u0026amp; Wengel, J. Oligodeoxynucleotides containing amide-linked LNA-type dinucleotides: synthesis and high-affinity nucleic acid hybridization. Chem. Commun., 530\u0026ndash;531 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeth, P.P. \u0026amp; Swayze, E.E. Oligomeric compounds comprising neutrally linked terminal bicyclic nucleosides PCT/US2009/039438 ISIS Pharmaceuticals WO/2009/124238 2009\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShivalingam, A., Tyburn, A.E.S., El-Sagheer, A.H. \u0026amp; Brown, T. Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation. J. Am. Chem. Soc. \u003cb\u003e139\u003c/b\u003e, 1575\u0026ndash;1583 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObika, S., Nakagawa, O., Hiroto, A., Hari, Y. \u0026amp; Imanishi, T. Synthesis and properties of a novel bridged nucleic acid with a P3\u0026prime; \u0026rarr; N5\u0026prime; phosphoramidate linkage, 5\u0026prime;-amino-2\u0026prime;,4\u0026prime;-BNA. Chem. Commun., 2202\u0026ndash;2203 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStork, G., Zhang, C., Gryaznov, S. \u0026amp; Schultz, R. Modified ollgonucleotides. Effect of 4 vs 5-atom chimeric internucleoside linkages on duplex stability. Tetrahedron Lett. \u003cb\u003e36\u003c/b\u003e, 6387\u0026ndash;6390 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmmenn, J., Altmann, K.-H. \u0026amp; Belluš, D. Aza-Claisen Rearrangement: Synthesis of 5\u0026prime;-branched 5\u0026prime;-aminothymidines. Helv. Chim. Acta \u003cb\u003e80\u003c/b\u003e, 1589\u0026ndash;1606 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoshkin, A.A., Fensholdt, J., Pfundheller, H.M. \u0026amp; Lomholt, C. A Simplified and Efficient Route to 2\u0026prime;-O, 4\u0026prime;-C-Methylene-Linked Bicyclic Ribonucleosides (Locked Nucleic Acid). J. Org. Chem. \u003cb\u003e66\u003c/b\u003e, 8504\u0026ndash;8512 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebreton, J., Waldner, A., Fritsch, V., Wolf, R.M. \u0026amp; De Mesmaeker, A. Comparison of two amides as backbone replacement of the phosphodiester linkage in oligodeoxynucleotides. Tetrahedron Lett. \u003cb\u003e35\u003c/b\u003e, 5225\u0026ndash;5228 (1994).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeterson, M.A. et al. Amide-Linked Ribonucleoside Dimers Derived from 5\u0026prime;-Amino-5\u0026prime;-deoxy- and 3\u0026prime;-(Carboxymethyl)-3\u0026prime;-deoxynucleoside Precursors(1). J. Org. Chem. \u003cb\u003e64\u003c/b\u003e, 8183\u0026ndash;8192 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKotikam, V. \u0026amp; Rozners, E. Concurrent Hydrogenation of Three Functional Groups Enables Synthesis of C3\u0026prime;-Homologated Nucleoside Amino Acids. Org. Lett. \u003cb\u003e19\u003c/b\u003e, 4122\u0026ndash;4125 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlbrecht, H., Jones, G. \u0026amp; Moffatt, J. 3'-Deoxy-3'-(dihydroxyphosphinylmethyl)nucleosides. Isophosphonate Analogs of Nucleoside 3'-Phosphates. J. Amer. Chem. Soc. \u003cb\u003e92\u003c/b\u003e, 5511\u0026ndash;5513 (1970).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArzel, L. et al. Synthesis of Ribonucleosidic Dimers with an Amide Linkage from d-Xylose. J. Org. Chem. \u003cb\u003e81\u003c/b\u003e, 10742\u0026ndash;10758 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVorbr\u0026uuml;ggen, H. Some recent trends and progress in nucleoside synthesis. Acta Biochim. Pol. \u003cb\u003e43\u003c/b\u003e, 25\u0026ndash;36 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrandsen, B.M., Hesser, A.R., Castner, M.A., Chandra, M. \u0026amp; Silverman, S.K. DNA-Catalyzed Hydrolysis of Esters and Aromatic Amides. J. Amer. Chem. Soc. \u003cb\u003e135\u003c/b\u003e, 16014\u0026ndash;16017 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKopka, M.L., Lavelle, L., Han, G.W., Ng, H.-L. \u0026amp; Dickerson, R.E. An Unusual Sugar Conformation in the Structure of an RNA/DNA Decamer of the Polypurine Tract May Affect Recognition by RNase H. J. Mol. Biol. \u003cb\u003e334\u003c/b\u003e, 653\u0026ndash;665 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang, S.-H., Cho, M.-J. \u0026amp; Kole, R. Up-Regulation of Luciferase Gene Expression with Antisense Oligonucleotides: Implications and Applications in Functional Assay Development. Biochemistry \u003cb\u003e37\u003c/b\u003e, 6235\u0026ndash;6239 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBailey, J.K., Shen, W., Liang, X.-H. \u0026amp; Crooke, S.T. Nucleic acid binding proteins affect the subcellular distribution of phosphorothioate antisense oligonucleotides. Nucleic Acids Res. \u003cb\u003e45\u003c/b\u003e, 10649\u0026ndash;10671 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRigo, F. et al. Synthetic oligonucleotides recruit ILF2/3 to RNA transcripts to modulate splicing. Nat. Chem. Biol. \u003cb\u003e8\u003c/b\u003e, 555\u0026ndash;561 (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1542733/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1542733/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOligonucleotides that target mRNA have great promise as therapeutic agents for life-threatening conditions but suffer from poor bioavailability, hence high cost. As currently untreatable diseases come within the reach of oligonucleotide therapies, new analogues are urgently needed to address this. With this in mind we have developed reduced-charge oligonucleotides containing artificial LNA-amide linkages with improved gymnotic cell uptake, RNA affinity, stability and potency. To construct such oligonucleotides, five LNA-amide monomers (A, T, C, 5mC and G), where the 3\u0026acute;-OH is replaced by an ethanoic acid group, were synthesised in good yield and used in solid-phase oligonucleotide synthesis to form amide linkages with high efficiency. The artificial backbone causes minimal structural deviation to the DNA:RNA duplex. These studies indicate that splice-switching oligonucleotides containing LNA-amide linkages and phosphorothioates display improved activity relative to oligonucleotides lacking amides, highlighting the therapeutic potential of this technology.\u003c/p\u003e","manuscriptTitle":"Oligonucleotide analogues with locked-amide linkages have therapeutic potential","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-05-04 18:58:35","doi":"10.21203/rs.3.rs-1542733/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"803e0358-fc59-4476-b642-03a4bc1f08aa","owner":[],"postedDate":"May 4th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2022-07-13T07:11:19+00:00","versionOfRecord":{"articleIdentity":"rs-1542733","link":"https://doi.org/10.1038/s41467-022-31636-2","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2022-07-12 04:00:00","publishedOnDateReadable":"July 12th, 2022"},"versionCreatedAt":"2022-05-04 18:58:35","video":"","vorDoi":"10.1038/s41467-022-31636-2","vorDoiUrl":"https://doi.org/10.1038/s41467-022-31636-2","workflowStages":[]},"version":"v1","identity":"rs-1542733","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1542733","identity":"rs-1542733","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}