Development and functional evaluation of a psoralen-conjugated nucleoside mimic for triplex-forming oligonucleotides

Abstract

Psoralen-conjugated triplex-forming oligonucleotides (Ps-TFOs) have been employed for the photodynamic regulation of gene expression by the photo-cross-linking of psoralen with the target DNA. However, stable triplex formation requires a consecutive purine base sequence in one strand of the target DNA duplexes. The pyrimidine-base interruption in the consecutive purine base sequence drastically decreases the thermodynamic stability of the corresponding triplex, which hampers the TFO application. Here, we propose a design of the Ps-TFO for stable triplex formation with target DNA sequences containing pyrimidine-base interruptions under physiological conditions. This Ps-TFO, named 1'(one)-psoralen-conjugated triplex-forming oligonucleotide (OPTO), incorporates a synthesized nucleoside mimic 1'-psoralen-conjugated deoxyribose to increase the thermodynamic stability of the corresponding triplex by the intercalation of psoralen. The triplex-forming abilities of the OPTO were successfully demonstrated in combination with LNA and 5-methylcytosine, indicating that the use of OPTO will expand the range of the target sequences of TFO for photodynamic gene regulation.

Fig. 1. Structural features of the triplex-forming oligonucleotides.

a The parallel triplex forms via a T·A:T triad and C + ·G:C triad. b Mismatched base pairs (T:A or C:G base pairs). A stable triplex formation with these pyrimidine base-interrupting sequences is challenging.

Fig. 2. The working hypothesis of this research.

The established structure of a psoralen-conjugated triplex-forming oligonucleotide (TFO) with the proposed design of (1’(one)-psoralen-conjugated triplex-forming oligonucleotide (OPTO)) is shown. The optimized orientation of psoralen in OPTO is better for intercalating psoralen into the target DNA.

Scheme 1

Synthesis of 1’-psoralen-conjugated deoxyribose phosphoramidite (12). Reagents and conditions: (i) trimethylsilyl cyanide (1.5 equiv), BF₃·Et₂O (0.3 equiv), CH₂Cl₂, RT, N₂, 1 d, 58% yield; (ii) 1. 28% NaOMe solution in Methanol (6 equiv), H₂O (3 equiv), Methanol, RT, 4 h; 2. strong acidic cation exchange resin No. 4 (8% cross-linking, 50-100 mesh, H type, 21 g), RT, 1 h, 68% yield; (iii) TBSCl (3 equiv), imidazole (3 equiv), CH₂Cl₂, RT, N₂, overnight, 71% yield; (iv) 1 M NaOH solution (1 equiv), MeOH/THF (1:1), RT, N₂, 0.5 h, 98% yield; (v) 5-aminopentanol (2 equiv), HBTU (2 equiv), triethylamine (4 equiv), CH₂Cl₂, RT, N₂, overnight, 76% yield; (vi) methanesulfonyl chloride (1.2 equiv), pyridine, RT, N₂, 3 h, 87% yield; (vii) 5-hydroxypsoralen (1.2 equiv), potassium carbonate (1.2 equiv), DMF, 60 ˚C, N₂, 3 h, 96% yield; (viii) 1 M solution of tetrabutylammonium fluoride in THF (2.4 equiv), THF, 60 ˚C, N₂, 2 h, 70% yield; (ix) 4, 4ʹ-dimethoxytrityl chloride (1.1 equiv), pyridine, RT, N₂, 1.5 h, 86% yield; (x) 1H-tetrazole (1.2 equiv), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphordiamidite (1.2 equiv), CH₂Cl₂, RT, N₂, 1.5 h, 87% yield.

Fig. 3. The UV-melting profile of the triplex of OPTO.

a sequences of the polypurine target (PPT) duplex (606-Py/606-Pu), TFO, and OPTO. b Structure of the 1’-psoralen-conjugated deoxyribose (P). c Normalized UV-melting curves of triplexes (duplex/TFO) and (duplex/OPTO) at pH 5.3. The final concentration of duplex and TFO was 3 μM each. The melting temperature (T_m) values are displayed as the mean ± standard deviation (s.d.) for n = 3 replicates.

Fig. 4. The UV-melting profiles of the triplex of L-OPTO.

a Structures of locked nucleic acid (LNA) and the conformational differences of the ribose moiety. b Sequences of the PPT duplex (606-Py/606-Pu), LNA-incorporated TFO (L-TFO), and LNA-incorporated OPTO (L-OPTO1–4). c Normalized UV-melting curves of triplexes (duplex/L-TFO) and (duplex/(L-OPTO1–4 = L1–4)) at pH 7.0 and the corresponding T_m values of the triplexes. The final concentration of duplex and TFO was 3 μM each. The T_m values are displayed as the mean ± s.d. for n = 3 replicates. d Non-denaturing polyacrylamide gel electrophoresis (Native PAGE) of triplexes (duplex/L-TFO) and (duplex/(L-OPTO1–4 = L1–4)) at pH 7.0 and 31 °C or 37 °C. The final concentration of duplex and TFO was 1 μM each. The bands were detected using SYBR^® Gold stain (Thermo Fisher Scientific, USA).

Fig. 5. The UV-melting profiles of the triplex of L-OPTO with varied sequences.

a Sequences of four PPT duplexes (Py-1/Pu-1, Py-2/Pu-2, Py-3/Pu-3, and Py-4/Pu-4), LNA-incorporated TFO (L-TFO2–5), and LNA-incorporated OPTO (L-OPTO5–8). b Normalized UV-melting curves of each triplex at pH 7.0 and their respective T_m values. The T_m values of L6-8 were determined by differential method from the region between the dotted line. The final concentration of duplex and TFO was 3 μM each. The T_m values are displayed as the mean ± s.d. for n = 3 replicates. c Native PAGE of the triplexes containing L-OPTO6–8 (L6–8) at pH 7.0 and 37 °C. The final concentration of duplex and TFO was 1 μM each. The bands were visualized using SYBR® Gold stain.

Fig. 6. The photo-cross-linking properties of L-OPTO.

a Photo-cross-linking of psoralen with the pyrimidine base and the sequence of the PPT duplexes. The correlation between the cross-linking efficiency and the number of psoralen molecules that were introduced into the TFO sequence (L-OPTO2-4) was examined. b The photo-irradiation (365 nm) was conducted at pH 7.0 and 37 °C. The final concentration of duplex and TFO was 1 μM each. The samples were collected at each irradiation time (0, 1, 5, 10, 15, 20, 25, and 30 s), and the product formation was analyzed by denaturing P. The 3’ end of the L-OPTOs was labeled with tetramethylrhodamine (TAMRA). The graphs show the quantification results of the photo-cross-linking efficiency of each sequence. The data are displayed as the mean ± s.d. for n = 3 replicates.