A solid-phase method for peptide–siRNA covalent conjugates based on click chemistry

Abstract

In order to prepare a new delivery system by chemical modification, peptide–siRNA conjugates have been obtained by a solid-phase click chemistry strategy, using an alkynyl nucleoside analogue “clicked” onto a peptide-derivatized CPG (Controlled Pore Glass) followed by oligonucleotides synthesis. The 3′-sense strand conjugate maintained good gene silencing activity, while that of the 3′-antisense strand conjugate decreased somewhat.

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Introduction

The phenomenon of RNA interference, a fundamental biological process which was discovered in the late 1990s, was very rapidly developed from a basic scientific discovery to a promising therapeutic approach.¹ Small interfering RNA (siRNA) has emerged as a new therapeutic class of drugs, and several clinical trials are currently in progress to develop siRNA for the treatment of various diseases.² Initial results suggested that siRNAs themselves were well-tolerated from a safety perspective.

Before the full potential of RNAi can be realized, methods are needed for delivering siRNAs safely, effectively and conveniently into the right tissues and cells. Development of an effective delivery vector is essential for RNAi therapy because of the difficulty of cellular uptake. Several viral and non-viral siRNA delivery systems have been studied deeply.³ Non-viral systems have advantages over viral vectors with regard to the convenience of complex formation, the ease of large-scale production, and the low risk of immunogenicity.⁴ Thus many chemical modifications have been used in the RNAi system for delivery in recent years.

Compared to synthetic vectors such as cationic polymers, branched dendrimers, and cationic liposomes, cell penetrating peptides (CPPs) represented a new promising approach and displayed advantage in the safety profile with low toxicity. CPPs are a novel type of membrane translocation agents. Many CPPs have been used to enhance the cellular uptake of oligonucleotides.⁵ While there are several applications using cell penetrating peptides in siRNA delivery, they are not very widely used. The first use of a CPP-mediated siRNA delivery was reported in 2003 by Divita et al.,⁶ in which peptide and siRNA formed a non-covalent complex. Focused on peptide–siRNA conjugation, we have sought to develop a synthetic method to covalently link the peptide with oligonucleotide motifs, aiming to provide an efficient and compact route to prepare these conjugates.

Results and discussion

Synthesis of peptide–siRNA conjugates

We report a solid-phase synthetic strategy to achieve the covalent conjugation of peptides and siRNAs through a 1,2,3-triazole linker. The sequence LALLAK (H-Leu Ala Leu Leu Ala Lys-OH) is a signal peptide mimic developed by Li and Chen,⁷ which they coupled to the 3′-end of antisense oligonucleotides by an amide bond. They found that it improved the permeability of antisense oligonucleotides through the cell membrane. We anticipate that this sequence should also enhance the permeability of siRNA. Therefore we uploaded this peptide step-wise using the standard Boc strategy to an amino-ON CPG support used for oligonucleotides synthesis (Scheme 1).

Scheme 1 Peptide derivatization of CPG. (i) Ref. 7; (ii) α-Fmoc-ε-Boc-Lys-OH, DCC, DMAP/DMF/DCM; and (iii) solid-phase peptide synthesis with Boc chemistry.

As the CPG is a non-swelling support, amino groups on its surface cannot be extended and therefore are not ideal for peptide synthesis. So that, the amino group was converted to the hydroxy group to enhance the reactivity of peptide synthesis and cleavage of conjugates from the solid phase carrier. After the derivatization, the peptide motif was cleaved and its sequence confirmed by MALDI-TOF-MS.

The 3′-terminal overhangs of siRNA usually are UU or TT, and chemical modification of them had no influence on the RNAi effect. Thus deoxyuridine was applied to get the 1,2,3-triazolelinker and as a part of overhangs. Both base (7) and sugar (3) modification building blocks were synthesized to achieve the above target. The 3′-alkynyl deoxyuridine (3), which has high cycloaddition activity with small azide derivatives, was synthesized in good yield from 2′-deoxyuridine via compound 2. The 5-modification deoxyuridine (7) was also synthesized from deoxyuridine.

Firstly, 5-hydroxymethyl derivate (4) was obtained by hydroxymethylation of deoxyuridine with paraformaldehyde. After chlorination by trimethylchlorosilane, sodium azide was used to give the azide derivative (5).⁸ Compound 6 was obtained by protecting 5′and 3′ hydroxyl groups of the sugar ring with DMTr-Cl and acetic anhydride respectively. Finally click reaction was employed to give the compound 7 containing a 1,2,3-triazole unit (Scheme 2).

Scheme 2 Synthesis of 3′-alkynyl deoxyuridine. (i) DMTrCl, Py; (ii) K₂CO₃, 3-bromopropyne, CH₃CN; (iii) 0.5 N KOH, (HCHO)_n; (iv) TMSCl, dioxane then NaN₃, DMF; (v) DMTrCl, Py, then Ac₂O, Py; (vi) CuI, DIPEA.

There are two strategies to load the modification nucleosides on the peptide derivatized CPG. For compound 3, using the “Click” idea, the terminal amino group of peptide derivatized CPG (CPG 1) was converted into azido group,⁹ which could construct the functional resin (CPG 2) by solid-phase click chemistry (Scheme 3). By importing a flexible C6 chain, the outreach of resin was improved and increased the collision probability between azide and alkynyl groups. Initially, azido compound (6) was used to react with peptide derived CPGs which have an alkynyl group, but the steric hindrance of DMT and the azide group located at the allylic position made the loading fail. While, compound 7 containing the 1,2,3-triazole unit was obtained in solution successfully.

Scheme 3 Solid-phase click reaction. (i) HOBt, HBTU, DIPEA, 6-azido-1-caproic acid; (ii) HOBt, HBTU, DIPEA, Boc-6-amino-1-caproic acid. 33% TFA/DCM; (iii) CuI, DIPEA, CH₃CN; and (iv) DIPEA, CH₃CN.

As an activated ester, compound 7 could be aminolysed under alkaline conditions, which formed CPG 3 (Scheme 3). The “click” efficiency and load of nucleoside can be determined easily by measuring the UV absorbance value of DMTr at 507 nm. The result showed that the load of CPG2 was suitable for oligonucleotides synthesis, while CPG3 had too low load to be used. Thus the functional resin CPG2 was used for the synthesis of peptide–oligonucleotide conjugates (a–c) on a DNA synthesizer with 1,2,3-triazole as a linker. After purification by HPLC, the desired conjugates prepared as described were >95% purity following HPLC analysis. MALDI-TOF-MS spectra unambiguously confirmed that the molecular weights of these final products were concordant with the calculated value of the designed sequence (Scheme 4). By this means, peptide (LALLAK)–RNA conjugates b and c were synthesized. For hybridization to form corresponding siRNA conjugates, naturally complementary RNA strands d (5′-UAA UAG AGA AAU UUC CCG AUU-3′) and e (5′-UCG GGA AAU UUC UCU AUU AUU-3′) were synthesized separately.

Scheme 4 Synthesis of conjugates. (i) Standard oligonucleotide synthesis; (ii) ammonium cleavage and deprotection; and (iii) HPLC purification. (a) Oligo = DNA: 5′-dT₉dU-3′; (b) Oligo = RNA: 5′-UCG GGA AAU UUC UCU AUU AUdU-3′; and (c) Oligo = RNA: 5′-UAA UAG AGA AAU UUC CCG AUdU-3′.

The duplex siRNAs I (e/d), II (b/d) and III (e/c) (Fig. 1) were generated by annealing with complementary sequences. Specifically, II (b/d) and III (e/c) have the same sequence only differing in the conjugation site: one is at the 3′-end of the passenger strand and the other is at the 3′-end of the guide strand. The thermal consequences of peptide conjugation were evaluated by UV melting experiments. The T_m value of conjugates II and III were 73.02 °C and 73.06 °C respectively. The unmodified siRNA I, hybridised by strands e and d, showed a T_m value of 74.00 °C. Thus, it is clear that the peptide moiety slightly reduces the siRNA duplex stability. It is possible that the peptide chains extend away from duplex, stretching the structure of conjugate position, and this may reduce the double-strand binding affinity, thereby reducing the T_m value.

Fig. 1 Structures of siRNA and peptide–siRNA conjugates used in this study.

Biological evaluation of peptide–siRNA conjugates

The duplex siRNAs were used to inhibit the expression of the cdc-2 mRNA. The inhibition efficiency of siRNA to target mRNA was detected by using the siQuant™ approach¹⁰ in HEK-293 cells. By means of the dual-luciferase test, residual activity of mRNA was determined to indicate the silencing effect, the smaller the value means the higher the silencing efficiency of siRNA. Under the same conditions as that for targeting sense mRNA, the conjugate II showed almost the same silencing activity (92.8%) compared with I as positive control (93.0%), illustrating that peptide modification on the passenger strand showed no discernible impact. By contrast, the guide strand conjugate III reduced the silencing efficiency to target mRNA to a certain extent (89.1%).

As we know that both strands of siRNA can be loaded into RISC and execute a knockdown effect of the corresponding target, this phenomenon may be due to the competitive effect between the two single strand loadings into RISC.¹¹

Interestingly, by comparing with unmodified siRNA I and antisense conjugate III, the sense strand 3′ terminal conjugate II decreased the silencing efficiency targeting antisense mRNA remarkably (Fig. 2, red bars). It suggested that 3′ terminal peptide modification could mitigate the off-target effect by sense strand loading onto RISC,¹² and also antisense conjugate III increased the silencing efficiency of sense strand targeting the antisense mRNA, which are consistent with the results of isonucleoside incorporated siRNAs (Fig. 2).¹³

Fig. 2 Gene silencing efficiency of siRNA I–III.

Experimental

General experimental

Analytical TLC was performed on precoated (250 μm) silica gel 60 F-254 plates from Merck. All plates were visualized by UV irradiation. Flash chromatography grade silica gel 60H (200–300 mesh) manufactured by QingDao Haiyang Chemical Company (China) was used for column chromatography. ¹H and ¹³C NMR spectra were recorded on JEOL JNMAL300 and Bruker ULTRASHIELD 400 plus instruments with TMS as an internal standard. ¹³C NMR spectra were calibrated relative to dioxane (d¼ 66.66 ppm) in a separate NMR tube. Chemical shifts (δ values) and coupling constants (J values) were given in parts per million and Hertz, respectively. ESI-TOF mass spectra were obtained on PE SCLEX QSTAR instruments, and the data are reported in m/z (intensity to 100%). MALDI-TOF mass analysis was performed on AXIMA, CFR plus MALDI-TOF instruments with 3-hydroxypicolinic acid or 2′,4′,6′-trihydroxy-acetophenone as a matrix. HPLC was carried out using a Waters instrument equipped with a UV detector and Phenomenex Clarity 10 μm Oligo-WAX Column 300 Å (150 × 10.0 mm) or 10 μm Oligo-WAX Column 300 Å (100 × 4.6 mm). All conjugates were freeze-dried using a Thermo MicroModulyo-230. All other used agents were marketed AP or CP agents.

5′-O-(4,4′-Dimethoxytrityl)-3′-O-(2-propynyl)-2′-deoxyuridine 3

Compound 2 (530 mg) and K₂CO₃ (150 mg) were placed in a dried round bottomed flask and anhydrous acetonitrile (10 mL) was added to the flask followed by 3-bromo-1-propyne (0.1 mL). The mixture was stirred for 10 h at 50 °C after which analysis by TLC indicated complete reaction. After filtration, the solvent was removed under reduced pressure. The residue was purified by flash column chromatography over silica gel using hexane and ethyl acetate (3 : 1) as eluant to give the final compound as a light-yellow solid in 88% yield (503 mg).

¹H NMR (300 MHz, CDCl₃): δ 7.80 (d, J = 8.1 Hz, 1H, H-6), 7.39–7.26 (m, 9H, Ar–H), 6.85 (d, J = 8.7 Hz, 4H, Ar–H), 6.33 (t, J = 6.3 Hz, 1H, H-5), 5.50 (d, J = 8.1 Hz, 1H, H-1′), 4.69 (d, J = 2.4 Hz, 2H, 3′-O-CH₂), 4.56 (s, 1H, NH), 4.02 (d, J = 3.3 Hz, 1H, H-4′), 3.80 (s, 6H, 2OCH₃), 3.49–3.45 (m, 2H, 5′-CH₂), 2.46–2.26 (m, 2H, 2′-CH₂), 2.17 (s, 1H, C≡CH), 1.84 (d, J = 3.9 Hz, 1H, H-3′). ¹³C NMR (100 MHz, DMSO-d₆): δ 29.7, 55.0, 63.3, 69.8, 73.0, 78.9, 85.2, 85.5, 85.8, 100.6, 113.2, 126.7, 127.7, 127.9, 135.2, 135.4, 139.4, 144.6, 149.7, 158.1, 160.8. ESI-MS: 569.3 (M + H⁺), 591.2 (M + Na⁺).

5-Hydroxymethyl-2′-deoxyuridine 4

To a solution of 2′-deoxyuridine (2.0 g) in 0.5 N KOH (30 mL), paraformaldehyde (2.6 g) was added. The mixture was stirred at 70 °C for 60 h. The solvent was removed under reduced pressure and the residue was purified by column chromatography using dichloromethane and methanol (10 : 1) as eluant to give the final compound as a white solid in 80% yield (1.8 g).

¹H NMR (300 MHz, DMSO-d₆): δ 11.31 (s, 1H, NH), 7.72 (s, 1H, H-6), 6.18 (t, J = 6.0 Hz, 1H, H-1′), 4.22 (s, 1H, H-4′), 4.12 (s, 2H, 5-CH₂), 3.76 (d, J = 3.0 Hz, 1H, H-3′), 3.54 (s, 2H, 5′-CH₂), 3.30 (d, J = 6.0 Hz, 1H, 3′-OH), 2.67 (t, J = 6.0 Hz, 1H, 5-OH), 2.08 (d, J = 6.0 Hz, 2H, 2′-CH₂), 1.02 (t, J = 6.0 Hz, 1H, 5′-OH). ESI-MS: 259.09 (M + H⁺), found 259.09 (M + H⁺).

5-Azidomethyl-2′-deoxyuridine 5

To a solution of compound 4 (4.0 g) in anhydrous dioxane (115 mL) under Ar, trimethyl chlorosilane (10 mL) was added. After stirring for 4 h at 60 °C, the solvent was removed under reduced pressure. The residue was dissolved in anhydrous N,N-dimethylformamide (45 mL) and sodium azide (3.0 g) was added. The mixture was stirred at 60 °C for 1 h. After filtration, the solvent was removed under reduced pressure. The residue was purified by column chromatography over silica gel using dichloromethane and methanol (10 : 1) as eluant to give the final compound as a yellow solid in 60% yield (2.6 g).

¹H NMR (300 MHz, DMSO-d₆): δ 11.59 (s, 1H, NH), 8.05 (s, 1H, H-6), 6.15 (t, J = 6.0 Hz, 1H, H-1′), 5.18 (d, J = 6.0 Hz, 2H, 5-CH₂), 4.24 (s, 1H, H-4′), 3.78 (s, 1H, H-3′), 3.58 (s, 2H, 5′-CH₂), 3.03 (dd, J = 6.0 Hz, 15.0 Hz 1H, 3′-OH), 2.11 (s, 2H, 2′-CH₂), 1.02 (t, J = 6.0 Hz, 1H, 5′-OH). ¹³C NMR (75 MHz, DMSO-d₆): δ 45.5, 46.9, 61.2, 70.2, 84.2, 87.5, 108.3, 139.9, 150.2, 162.9. ESI-MS: 284.1 (M + H⁺), found 284.1 (M + H⁺).

5-Azidomethyl-5′-O-(4,4′-dimethoxytrityl)-3′-acetoxy-2′-deoxyuridine 6

To a solution of compound 5 (840 mg) in anhydrous pyridine (8 mL), a solution of DMTrCl (1.32 g, 1.3 eq.) in anhydrous pyridine (6 mL) was added dropwise. The mixture was stirred at 0 °C for 2 h and continued stirring at room temperature overnight. The solvent was removed under reduced pressure. The residue was dissolved in anhydrous pyridine (25 mL), and acetic anhydride (1 mL) was added. After stirring for 1.5 h at room temperature, the solvent was removed under reduced pressure, and the residue was purified by column chromatography over silica gel using petroleum ether (boiling range 60–90 °C) and ethyl acetate (1 : 1) as eluant to give the final compound as a light-yellow solid in 84% yield (1.56 g).

¹H NMR (300 MHz, CDCl₃): δ 7.84 (s, 1H, H-6), 7.38–7.24 (m, 9H, Ar–H), 6.86 (d, J = 8.7 Hz, 4H, Ar–H), 6.46–6.41 (m, 1H, NH), 5.48 (d, J = 5.1 Hz, 1H, H-1′), 4.15 (s, 1H, H-4′), 3.80 (s, 6H, 2-OCH₃), 3.47 (s, 2H, 5-CH₂), 3.23–3.19 (d, J = 13.5 Hz, 1H, H-3′), 2.59–2.42 (m, 4H, 5′-CH₂ and 2′-CH₂), 2.10 (s, 3H, 3′-COCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 8.1, 20.9, 38.2, 46.4, 52.9, 55.2, 63.5, 75.4, 84.2, 84.5, 110.3, 113.3, 127.3, 128.1, 130.1, 134.9, 135.1, 144.0, 150.0, 158.8. ESI-MS: 650.22 (M + Na⁺), found 650.25 (M + Na⁺).

5-(4′-Methoxycarbonyl-1′,2′,3′-triazole-1′-methyl)-5′-O-(4,4′-dimethoxytrityl)-3′-acetoxy-2′-deoxyuridine 7

To a solution of cuprous iodide (19 mg) and DIPEA (0.2 mL) in anhydrous acetonitrile (6 mL), methyl propiolate (0.1 mL) was added slowly at 0 °C under Ar. The mixture was stirred at 0 °C for 0.5 h and returned to room temperature. Compound 6 (627 mg) in anhydrous acetonitrile (6 mL) was added to the mixture slowly. The mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and the residue was purified by column chromatography over silica gel using dichloromethane and methanol (100 : 1) as eluant to give the final compound as a light-yellow solid in 84% yield (600 mg).

¹H NMR (300 MHz, CDCl₃): δ 8.19 (s, 1H, =CH–), 8.12 (s, 1H, H-6), 7.41–7.22 (m, 9H, Ar–H), 6.86 (d, J = 8.4 Hz, 4H, Ar–H), 6.42–6.38 (m, 1H, NH), 5.53 (d, J = 5.4 Hz, 1H, H-1′), 4.56–4.51 (d, J = 14.7 Hz, 1H, H-4′), 4.17 (s, 2H, 5-CH₂), 3.93 (s, 3H, COOCH₃), 3.78 (s, 6H, 2OCH₃), 3.49 (dd, J = 7.2, 14.4 Hz, 2H, 2′-CH₂), 2.60–2.47 (m, 2H, 5′-CH₂), 2.10 (s, 3H, 3′-COCH₃). ¹³C NMR (100 MHz, CDCl₃): δ 20.9, 22.7, 29.7, 30.0, 31.9, 38.4, 46.5, 52.3, 55.3, 62.4, 75.0, 85.8, 86.3, 108.1, 113.2, 127.1, 127.7, 127.8, 128.6, 129.1, 139.5, 141.3, 147.3, 158.6, 170.5. ESI-MS: 734.2 (M + H⁺), found 734.3 (M + Na⁺).

Synthesis of CPG 1

(a) Loading of the first amino acid on CPG. The CPG, obtained by hydroxylation of amino-ON CPG (PE product, pore size 500 Å, 0.5 g, amino group loaded: 0.22 mmol g⁻¹), was drained and washed with dichloromethane (DCM), methanol, and DCM and then reacted with N-α-Fmoc-N-ε-Boc-L-lysine (57.4 mg, 0.11 mmol) and 4-dimethylaminopyridine (DMAP) (3 mg) in 2 mL of dimethylformamide (DMF) and DCM (3 : 1, v/v) for 2 min. To the mixture was added 0.2 mL of dicyclohexylcarbodiimide (DCC) (1 M), and the reaction was performed at 25 °C for 24 h. After the sample was washed with DCM and methanol, the CPG was treated with acetic anhydride (Ac₂O) and pyridine (Py) in DCM (3 mL, 1 : 1 : 2, v/v) for 20 min to protect the residual hydroxyl groups. The CPG was washed with DCM and methanol alternately and finally by DCM.

(b) Elongation of peptide. The Boc group at the first amino acid on the resulted CPG was removed by treatment with 33% TFA (trifluoroacetic acid) in DCM (v/v) at room temperature for 30 min. The CPG was washed (DCM and MeOH) and neutralized (10% triethylamine in DCM, v/v) for 10 min. Further peptide synthesis was carried out on the manual peptide synthesizer using standard Boc chemistry. The coupling reaction was performed sequentially using a 2.5-fold molar excess of N-α-Boc protected L-amino acids (A, L, L, A and L) and DCC with the reaction concentration of 0.1 M in DCM at room temperature for 2 h. The extent of the condensation was determined by ninhydrin assay. The retained amino groups were acetylated as before. The CPG was washed with DCM and methanol alternately and finally by DCM. To determine the component of the peptide, partial amount of the resulted CPG was treated by 33% TFA in DCM (v/v) followed by concentrated ammonium hydroxide at 55 °C for 14 h. After the sample was purified on HPLC, the peptide was identified by MALDI-TOF-MS. MALDI-TOF-MS m/z: 627.15 [M]⁺, 649.12 [M + Na]⁺, calculated for C₃₀H₅₇N₇O₇: m/z: 627.43.

Synthesis of CPG 2

(a) Introduction of azido group on CPG 1. The Boc group at the end amino acid on CPG 1 was removed by treatment with 33% TFA in DCM (v/v). The CPG was washed and neutralized. Then it was reacted with 5-fold molar excess of 6-azido hexanoic acid and DCC with the reaction concentration of 0.1 M in DCM at room temperature for 8 h. The extent of condensation was determined by ninhydrin assay. The retained amino groups were acetylated as before. The CPG was washed and dried in a vacuum.

(b) Solid phase click chemistry with compound 3. A mixture of 10-fold molar excess of compound 3, 10-fold DIPEA and 1-fold CuI in anhydrous acetonitrile (10 mL) with azido CPG (prepared above) was shaken at room temperature for 12 h to construct CPG 2. The CPG was washed with DCM and methanol alternately and finally by DCM. The calculated load of nucleoside on CPG 2 was 142 μmol g⁻¹ by measuring the UV absorbance value of DMTr at 507 nm (ε = 66 500) after being treated with 5 mL of 3% dichloroacetic acid (DCA) in DCM (v/v).

Synthesis of CPG 3

(a) Introduction of 6-amino-1-caproic acid on CPG 1. A mixture of 2.5-fold molar excess of Boc-6-amino-1-caproic acid, 2.5-fold HBTU and 2.5-fold HOBt was dissolved in DMF (5 mL) with 3.0-fold DIPEA. The mixture was shaken with CPG 1 (prepared above) at room temperature for 6 h. The CPG was washed alternately with DCM and methanol and finally by DCM. The extent of the condensation was determined by ninhydrin assay. The Boc group on CPG was removed by treatment with 33% TFA in DCM (v/v), and the CPG was washed and neutralized.

(b) Solid phase reaction with compound 7. A mixture of 10-fold molar excess of compound 7, 10-fold DIPEA in anhydrous acetonitrile (10 mL) with CPG (prepared above) was shaken at room temperature for 12 h to construct CPG 3. The CPG was washed with DCM and methanol alternately and finally by DCM. The calculated load of nucleoside on CPG 3 was 15 μmol g⁻¹ by measuring the UV absorbance value of DMTr at 507 nm (ε = 66 500) after being treated with 3% dichloroacetic acid (DCA, 5 mL) in DCM (v/v).

General synthesis of conjugate a–c on CPG 2¹⁴

A total of 20 mg of the resultant CPG obtained from the previous reaction served as the solid phase support and submitted to the cartridge of the Applied Biosystem 381 DNA synthesizer for the synthesis of the desired sequence of oligonucleotides. The reaction cycle was carried out in 2 μM scale using 2′-TBDMS (tert-butyl dimethylsilyl)-ribonucleoside phosphoramidites as coupling blocks, and the procedures were set in compliance with the protocol recommended except for a prolonged 600 s of the coupling wait step with the nucleoside monomer. The yield of each coupling reaction is 97–99%. The reaction was stopped at DMT-off. Normal cleavage and deprotection were completed with aqueous ammonia to obtain the peptide conjugated oligonucleotides. For RNA conjugates, further removal of TBDMS groups was carried out by adding 1 mL of TBAF (tetrabutylammonium fluoride solution 1 M in THF) to the dried conjugates and shaking for 6 h at room temperature followed by adding 1 mL of 1 M Tris (tris-(hydroxymethyl)-aminomethane) buffer (pH = 8.0) to quench the reaction. After desalting by Sephadex G-25 column (50% acetonitrile in water), these conjugates were purified by HPLC using a Oligo-WAX column (Phenomenex, 300 Å, 10 μm, 150 × 10.0 mm). The gradient was 0–45% B in 45 min (mobile phase A is 20 mM Tris–HCl with 10% CH₃CN (pH = 8.0), and mobile phase B is 2 M NaCl in A) with 3.0 mL min⁻¹ flow. The purified conjugates were re-desalted again through the G-25 Column. MALDI-TOF-MS analysis of a (5′-dT₉dU-3′-LALLAK) (m/z: 3771 [M + H]⁺; found 3779), b (5′-UCG GGA AAU UUC UCU AUU AUdU-3′-LALLAK) (m/z: 7408 [M + Na]⁺; found 7411) and c (5′-UAA UAG AGA AAU UUC CCG AUdU-3′-LALLAK) (m/z: 7477 [M + Na]⁺; found 7473).

General synthesis of RNA d and e

Instead of CPG 2, U-CPG served as the solid phase support to synthesize the unmodified RNA oligonucleotides by a similar process. Cleavage, deprotection and purification were done following the procedures described above. MALDI-TOF-MS analysis of d (5′-UAAUAGAGAAAUUUCCCGAUU-3′) (m/z: 6665 [M + H]⁺; found 6664) and e (5′-UCGGGAAAUUUCUCUAUUAUU-3′) (m/z: 6619 [M + Na]⁺; found 6620).

Conclusions

In summary, we have developed a concise route to synthesise peptide–siRNA conjugates by the solid-phase click chemistry strategy. With this strategy, peptides and siRNAs can be conjugated easily and efficiently without regarding the difference between sequences. Biological assay results showed that these kinds of peptide–siRNA conjugates retain their gene silencing efficiency and the off-target effect of the sense strand 3′ terminal conjugate is reduced due to more difficult loading onto RISC itself. Although much work is necessary to improve the membrane permeability of the conjugates, especially further optimization of the peptide sequence, a new strategy has been explored to enhance their biological properties in the future.

Acknowledgements

We thank Prof. Zi-Cai Liang, Quan Du and Fan Yi in Peking University for providing siRNA sequence and assistance in biological experiments. This work was supported by the National Natural Science Foundation of China (grant no. 20932001), the Ministry of Science and Technology of China (grant no. 2009ZX09503).

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14. To minimize RNase degradation of the oligoribonucleotide, wearing gloves and sterile materials were necessary during the purification, and all used water was treated by DEPC.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2md00198e

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