Locked nucleic acid (LNA) enhances binding affinity of triazole-linked DNA towards RNA† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7cc05159j

LNA improves the RNA-binding affinity and enzymatic stability of triazole-linked DNA.


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Nucleoside 5 1 (2.00 g, 3.50 mmol) was co-evaporated with anhydrous THF (3 × 15 mL) and redissolved in anhydrous THF (24 mL). The solution was cooled to 0 °C and NaH (60% suspension in mineral oil, 0.348 g, 14.5 mmol) was added in portions over 5 min. The reaction mixture was stirred on ice for 30 min and at room temperature for 1 h. Propargyl bromide (0.374 mL, 4.20 mmol) was added at 0 °C and the reaction was stirred on ice for 30 min and at room temperature for 16 h. Solvent was removed at reduced pressure and the residue was dissolved in EtOAc (100 mL) and washed with brine (2 × 50 mL). The organic phase was dried (Na 2 SO 4 ) and concentrated and the resulting crude was purified using column chromatography (EtOAc in hexane, 10% to 80%, v/v) to obtain compound 6 (1.68 g, 2.75 mmol, 79%) as a white foam.

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Nucleoside 6 (0.408 g, 0.668 mmol) was co-evaporated with anhydrous pyridine (3 × 10 mL) and redissolved in anhydrous pyridine (5 mL Amino-SynBase resin 500/100 (Link Technologies, Glasglow, UK) (500 Å pore size, loading 69 μmol/g, 4.06 g, 0.28 mmol of amine) was activated using 3% solution of trichloroacetic acid in CH 2 Cl 2 for 3 h in a stoppered glass vessel fitted with a sinter and tap. The solvents were removed by filtration and the support was washed with triethylamine:diisopropylethylamine (9:1), CH 2 Cl 2 , and diethyl ether. The support was dried under vacuum for 1 h and re-suspended in anhydrous pyridine (10 mL). A solution of succinic anhydride (0.813 g, 8.13 mmol) and DMAP (160 mg, 1.3 mmol) in anhydrous pyridine (5 mL) was added and the vessel was rotated at room temperature for 20 h. The solvents were removed by filtration, and the support was washed with pyridine, CH 2 Cl 2 , and diethyl ether and dried under high vacuum for 1 h. 500 mg of the activated resin was taken forward and soaked in 1 mL of anhydrous pyridine for 10 min. Diisopropyl carbodiimide (DIC) (93 μL, 0.60 mmol), 1hydroxybenzotriazole (HOBT) (93 μL, 0.69 mmol), and compound 7 (86 mg, 0.14 mmol) were added to the reaction vessel, and the vessel was rotated for 20 h at room temperature. Pentachlorophenol (45 mg, 0.17 mmol) was added, and the vessel was rotated for an additional 3 h. The solvents were removed by filtration, and the support was washed with pyridine, CH 2 Cl 2 , and diethyl ether. Piperdine (10% in DMF, 2 mL) was added and the vessel was rotated for 5 min at room temperature. The solvent was removed by filtration and the support was washed with CH 2 Cl 2 and diethyl ether. Capping reagent (oligonucleotide synthesis grade, acetic anhydride/pyridine/THF:N-methylimidazole in THF, 1:1, 2 mL) was added and the vessel was rotated at room temperature for 1 h. The solvent was removed by filtration, and the resin was washed with pyridine, CH 2 Cl 2 , and diethyl ether and dried under high vacuum overnight. Loading of nucleoside 7 on the support determined by cleaving the DMT group and was found to be 26 μmol/g.

Synthesis of DNA oligonucleotides
Standard DNA phosphoramidites, solid supports and reagents were purchased from Link Technologies and Applied Biosystems. LNA phosphoramidites were obtained from Exiqon. Automated solid phase synthesis of oligonucleotides (trityl off) was performed on an Applied Biosystems 394 synthesiser. Synthesis was performed on 1.0 µmole scale involving cycles of acidcatalyzed detritylation, coupling, capping, and iodine oxidation. Standard DNA phosphoramidites were coupled for 60 s while extended coupling time of 10 min was used for LNA phosphoramidites. Coupling efficiencies and overall synthesis yields were determined by the inbuilt automated trityl cation conductivity monitoring facility and were ≥98.0% in all cases. The oligonucleotides were then cleaved from the solid support and protecting groups from the nucleobase and backbone were removed by exposure to concentrated aqueous ammonium hydroxide for 60 min at room temperature followed by heating in a sealed tube for 5 h at 55 °C.

Synthesis of RNA oligonucleotides
2'-TBS protected RNA phosphoramidite monomers with t-butylphenoxyacetyl protection of the A, G and C nucleobases were used to assemble RNA oligonucleotides. Benzylthiotetrazole (BTT) was used as the coupling agent, t-butylphenoxyacetic anhydride as the capping agent and 0.1 M iodine as the oxidizing agent (Sigma-Aldrich). Coupling time of 10 min was used and coupling efficiencies of >97% were obtained. Cleavage of oligonucleotides from the solid support and protecting groups from the nucleobase and backbone were removed by exposure to concentrated aqueous ammonia/ethanol (3/1 v/v) for 2 h at room temperature followed by heating in a sealed tube for 2 h at 55°C.

Removal of 2'-TBS protection of RNA oligonucleotides
After cleavage from the solid support and removal of the protecting groups from the nucleobases and phosphodiesters in ammonia/ethanol as described above, oligonucleotides were concentrated to a small volume in vacuo, transferred to 15 mL plastic tubes and freeze dried (lyophilised). The residue was dissolved in DMSO (300 μL) and triethylamine trihydrofluoride (300 μL) was added after which the reaction mixtures were kept at 65 °C for 2.5 h. Sodium acetate (3 M, 50 μL) and butanol (3 mL) were added with vortexing and the samples were kept at −80 °C for 30 min then centrifuged at 13,000 rpm at 4 °C for 10 min. The supernatant was decanted and the precipitate was washed twice with ethanol (0.75 mL) then dried under vacuum.

Purification of oligonucleotides (DNA or RNA)
The fully deprotected oligonucleotides were then purified by reverse-phase high performance liquid chromatography (HPLC) on a Gilson system using a Luna 10 μm C8(2) 100 Å pore Phenomenex column (250 × 10 mm) with a gradient of acetonitrile in triethylammonium bicarbonate (TEAB) over 20 min at a flow rate of 4 mL per minute. Buffer A: 0.1 M TEAB, pH 7.5; buffer B: 0.1 M TEAB, pH 7.5, with 50% acetonitrile were used. Elution was monitored by UV absorption between 260-295 nm.

Synthesis of 5'-azide modified oligonucleotides
Trityl off oligonucleotides were assembled at 1.0 μmole scale and were treated with a 0.5 M solution of methyltriphenoxyphosphonium iodide in DMF (1.0 mL) while attached to the solid support in a synthesis column. 3 The solution was periodically passed through the column using two 1 mL syringes for 20 min at room temperature. The resin was then washed several times with DMF. In a separate vessel 50 mg of sodium azide was taken up in 1 mL DMF and heated to 70 °C for 10 min. The mixture was allowed to cool to room temperature and the supernatant was passed back and forth through the synthesis column using two 1 mL syringes. 4 The synthesis column was left at 55 °C for 5 h and during this time the solution was occasionally passed back and forth. The column was then washed with DMF followed by acetonitrile and dried by the passage of a stream of argon. The resultant 5´azide oligonucleotide was cleaved from solid support and deprotected by exposure to concentrated aqueous ammonium hydroxide for 60 min at room temperature followed by heating in a sealed tube for 5 h at 55 °C and purified as described above. Purified oligonucleotides were then characterised by mass spectrometry (Table S1).

Synthesis of 13-mer oligonucleotides incorporating a single triazole linkage
Representative Procedure: A mixture of 5´-azide oligonucleotide (130 nm) and 3´-alkyne oligonucleotide (100 nm) was freeze dried and re-dissolved in milli-Q water (250 µL). The solution was flushed with a stream of argon and to this was added an aqueous solution of CuSO 4 (20 µL, 100 mM), an aqueous solution of sodium ascorbate (40 µL, 500 mM), and tris-hydroxypropyltriazole ligand 5 (5 mg). The resulting mixture was degassed with a stream of argon and left at room temperature for 2 h with occasional shaking. Reagents were then removed by NAP-10 gel-filtration and the ligated triazole oligonucleotide was purified by HPLC (as described above) and characterized by mass spectrometry (Table S1).

Synthesis of 13-mer oligonucleotides incorporating two triazole linkages
Representative Procedure: A 5´-azide oligonucleotide, a 3´-alkyne oligonucleotide, a 5´-azide-3´alkyne oligonucleotide and a splint (40 nm each) were mixed with NaCl (200 µL, 3 M). Milli-Q water was added to raise the total volume to 1940 µL. The mixture was annealed by heating to 80 °C and then cooling slowly to room temperature. The content was then kept at 4 °C for 1 h. CuSO 4 (aqueous, 20 µL, 100 mM), sodium ascorbate (aqueous, 40 µL, 500 mM), and trishydroxypropyltriazole ligand 5 (4 mg) were added. Thus a final concentration of 20 µM of each oligo in 300 mM NaCl and a final volume of 2 mL was obtained. The reaction mixture was left at 4 °C for 3 h and then at room temperature for 1 h. Reagents were then removed by NAP-10 gel-filtration and the ligated triazole oligonucleotide was purified by denaturing 20% polyacrylamide gel electrophoresis and characterized by mass spectrometry (Table S1). Splint used: 5´-dTTTTTT GCTAGAGAAGTCG TTTTTT (For ON8 and ON9), 5´-dTTTTTTGCTGGAGAGGTCGTTTTTT (for ON13 and ON14).

Synthesis of an 81-mer template incorporating a single LNA-triazole linkage
ON32 and ON18 (Table S1, 70 nm of each) and a splint (70 nm) were mixed with NaCl (200 µL, 3 M) and total volume was brought to 1940 µL by the addition of milli-Q water. The mixture was annealed by heating to 80 °C and then cooling slowly to room temperature. CuSO 4 (aqueous, 20 µL, 100 mM), sodium ascorbate (aqueous, 40 µL, 500 mM), and tris-hydroxypropyltriazole ligand 5 (4 mg) were added. The reaction mixture was left at room temperature for 3 h. Reagents were then removed by NAP-10 gel-filtration and the ligated triazole oligonucleotide was purified by denaturing 12% polyacrylamide gel electrophoresis, and characterized by mass spectrometry (ON15, table S1). Splint used: 5´-dTGTGTGCTAGCGATCTTA.   Table 1 (main manuscript).   Table S2.   Figure S3: Representative melting curves for duplexes incorporating two triazole linkages ( Me C-T steps, left against DNA target and right against RNA target). For sequences see Table 2 (main manuscript). Table S4. Thermal melting (T m ) data for duplexes incorporating two triazole linkages ( Me C-Me C steps).  Table S2 footnote. DNA target: 5´-dGCT GGA GAG GTC G, RNA target: 5´-rG CUA GAG AAG UC G | 11 |  Figure S4: Representative melting curves for duplexes incorporating two triazole linkages ( Me C-Me C steps, left against DNA target and right against RNA target). For sequences see Table S4.

CD spectroscopy
CD spectra (200−340 nm) were recorded on a Chirscan Plus spectropolarimeter using a Quartz optical cells with a path length of 3.0 mm. Scans were performed at 20 °C using a step size of 0.5 nm, a time per point of 1.0 s and a bandwidth of 2 nm, and the average of four scans is presented. Samples from UV melting studies (3 µM of each oligonucleotide in a 10 mM phosphate buffer containing 200 mM NaCl at pH 7.0) were used directly and were annealed by heating to 85 °C and then slowly cooled to 20 °C prior to recording CD spectrum. The average trace was smoothed (20 points) using in built software. A CD spectrum of only buffer was also recorded and was subtracted from the collected data. Finally, spectra were baseline-corrected using the offset at 340 nm.

Linear copying of an 81-mer template incorporating a single LNA-triazole linkage
A reaction mixture was prepared by mixing 10 µL of 10 X NEB buffer 2* in a total reaction volume of 100 µL with template, primer or template + primer (110 pmol of each), 0.2 mM dNTP and 1.0 µL of DNA polymerase 1, Large Klenow fragment (5u/µL). Reaction mixture was left at 37°C for 2.5 h. Phenol:chloroform:isoamyl alcohol (25:24:1, v/v) solution (100 µL) was added and mixture was vortexed for 30 seconds, centrifuged for 5 min at 5000 rpm. Aqueous phase was collected and sodium acetate (10 μl, 3 M, pH 5.2) and ethanol (330 µL) were added. The mixture was left at −80 °C for 4 h and then centrifuged (13000 rpm) for 20 min at 4 °C. The supernatant was removed and the resulting pellet was dissolved in 20 µL H 2 O. 10 µL sample was used for mass and another 10 µL was analysed by denaturing 10% polyacrylamide gel electrophoresis ( Figure S8). Similar gels were obtained when reaction mixture was directly (prior to precipitation) loaded on the gel. Incubation of reaction mixture for 1.5 h showed truncated product in addition to full length product presumably stalling the reaction at the triazole step. The product was analysed by mass spectrometry.
*(10 X NEB buffer2 was supplied with the enzyme).1 X NEB buffer 2 = 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 , 1 mM DTT (pH 7.9 at 25 °C). Figure S8: 10% denaturing polyacrylamide gel from linear copying reaction. Lane 1; Linear copying reaction using modified template (ON15) 5´-dGCA TTC GAG CAA CGT AAG ATC G Me CtT L AGC ACA CAA TCT CAC ACT CTG GAA TTC ACA CTG ACA ATA CTG CCG ACA CAC ATA ACC where t represent triazole linkage and T L is LNA thymidine. Lane 2; Linear copying reaction using unmodified template 5´-dACGTTAGCACGAAGAGGCATCTTAGCACACAATCTCACACTCTGGAATTCACACTGACAATACTCGCGAACACACCCAAT. Lane 3; negative control: linear copying reaction using modified template without enzyme. For modified template: Full length product mass; found 26025, calc. 26025. A relatively small peak at 26337 (full length + A) was also observed. For unmodified template: Full length product mass; found 25695, calc. 25695. No M+A product was observed for control. Primer used: 5´-dFTGGTTATGTGTGTCGGCAG (for modified template), 5´-dFTATTGGGTGTGTTCGCGAG (for unmodified template), F is amidohexylfuorescein.

PCR of an 81-mer template incorporating a single LNA-triazole linkage
PCR amplification of the modified template (ON15) was achieved using GoTaq DNA polymerase. 10 µL of 5X buffer (Promega gree PCR buffer) was used in a total reaction volume of 50 µL with 12.5 ng of the DNA template, 0.5 µM of each primer, 0.2 mM dNTP and 1.25 unit of GoTaq polymerase. PCR cyclic conditions: 95 °C (initial denaturation) for 2 min then 3 cycles of 95 °C (denaturation) for 15 s, 54 °C (annealing) for 20 s and 72 °C extension for 5 min. Next 20 cycles 95 °C (denaturation) for 15 s, 54 °C (annealing) for 20 s and 72 °C extension for 30 s. This was followed by leaving the PCR reaction mixture at 72 °C for 5 min. The PCR amplicon was analysed by loading onto 2% agarose gel, and was precipitated following the procedure described for linear copying for mass analysis. Primers used: 5´ -dGCATTCGAGCAACGTAAG, 5´-dGGTTATGTGTGTCGGCAG (for modified 3