Remarkable acceleration of a DNA/RNA inter-strand functionality transfer reaction to modify a cytosine residue: the proximity effect via complexation with a metal cation

Modified nucleosides in natural RNA molecules are essential for their functions. Non-natural nucleoside analogues have been introduced into RNA to manipulate its structure and function. We have recently developed a new strategy for the in situ modification of RNA based on the functionality transfer reaction between an oligodeoxynucleotide probe and an RNA substrate. 2′-Deoxy-6-thioguanosine (6-thio-dG) was used as the platform to anchor the transfer group. In this study, a pyridinyl vinyl ketone moiety was newly designed as the transfer group with the expectation that a metal cation would form a chelate complex with the pyridinyl-2-keto group. It was demonstrated that the (E)-pyridinyl vinyl keto group was efficiently and specifically transferred to the 4-amino group of the opposing cytosine in RNA in the presence of NiCl2 with more than 200-fold accelerated rate compared with the previous system with the use of the diketo transfer group. Detailed mechanistic studies suggested that NiCl2 forms a bridging complex between the pyridinyl keto moiety and the N7 of the purine residue neighboring the cytosine residue of the RNA substrate to bring the groups in close proximity.

Scheme S2. The functionality transfer reaction using FT-ODN-S1 and RNA1, and the click reaction with biotin-N 3 or FAM-N 3 .

Experimental
Preliminary experiments using FT-ODN (1) prepared using the pyridinyl ethynyl keto derivative (5). The modification of 6-thio position of ODN (1) was performed using 100 M of ODN1 and 500 M of the alkylating agent (5) in 25 mM carbonate buffer at pH 10 and r.t. for 10 min. After dilution of the mixture, the transfer reaction was performed using 6 M of FT-ODN1, 5 M of RNA1, 50 mM HEPES buffer, 100 mM NaCl, 0.6 M NiCl 2 at pH 7.4 and 37 °C. The reaction progress of the modification of ODN1 and the transfer reaction to RNA1 (rC) were followed by HPLC ( Figure S1). HPLC conditions; column: SHISEIDO C18, 4.6 x 250 mm, solvents, A: 0.1M TEAA, B: CH 3 CN, B 10 % to 30 % /20 min, 30 % to 100 % /25 min, linear gradient; flow rate at 1.0 ml/min, UV monitored at 254 nm.

Confirmation of the modified position of RNA1
The modified RNA1 was isolated and subjected to MS/MS analysis using the following conditions. An Acquity UPLC H-Class TUV system (Waters, Milford, MA, USA) fitted with an Acquity BEH C18 column  Figure S4, clearly indicating that rC is modified as expected.

Determination of the structure of the product of the functionality transfer reaction
To determine the structure of the modified cytidine, the transfer reaction was performed using the corresponding DNA1, 5' AGAAAGGAGAA-C-AAAG., in which rC represents the target dC. The reaction was performed using 15 M of (E)-FT-ODN1 and 10 M of DNA1 in the buffer 50 mM HEPES and 100 mM NaCl, 1 mM NiCl 2 at pH 7 and 37°C. The modified DNA substrate was purified, S8 freeze-dried, and subjected to reduction in a carbonate buffer (25 mM, pH 10) containing 100 mM NaBH 4 for 30 min at room temperature. The reaction mixture was neutralized with acetic acid and purified by HPLC. The reduced DNA substrate was diluted with ten-times diluted BAP buffer, followed by the addition of bacterial alkaline phosphatase (BAP, 0.05 u/L), nuclease P1 (0.08 u/L) and venom phosphodiesterase (VPDE, 0.01 u/L). The mixture was incubated for 60 min at 37 °C, and analyzed by HPLC using the following conditions ( Figure S5). HPLC conditions: column, SHISEIDO CAPCELL PAK C18, TYPE MG; flow rate: 1 mL/min; solvent A = 50 mM HCOONH 4 , solvent B = CH 3 CN, 10 % to 55 % /20 min, 55 % to 100 % /25 min, linear gradient, monitored at 254 nm. The peak corresponding to the modified dC was confirmed by ESI-MS and comparison by HPLC co-injection with the authentic sample ( Figure S5).

3-Amino-1-(pyridin-2-yl)propan-1-ol (S3-2)
LiAlH 4 (657 mg, 17.11 mmol) was added into a solution of the above product (500 mg, 3.42 mmol) in THF (30 mL) at 0 °C under an argon atmosphere. The reaction mixture was heated to 80 °C under reflux. After 4 h, the reaction mixture was cooled to 0 °C, followed by the addition of water (3 mL) and 10 % aqueous NaOH (1.5 mL). The resulting precipitates were filtrated through a Celite pad and the filtrate was evaporated to dryness to give S3-2 as a brown foam (494 mg). The product was used for next step without further purification.

Kinetic analysis of the functionality transfer reaction using RNA1(rC) and (E)-FT-ODN1
The reaction within the DNA/RNA duplex was analyzed as the first-order reaction using the initial duplex concentration of 4.5 M as the reactive duplex formed with (E)-FT-ODN1, and the rest (0.5 M) as the nonreactive one formed with (Z)-FT-ODN1. The HPLC peak of rC-modified RNA1 was quantified and the half-life (t 1/2 s) of the reaction was obtained, then the first-order rate constant (k 1 ) was calculated by the equation (1). The k 1 values were obtained at the different temperature (15,20,25,30 and 35°C) and in the presence of different concentrations of NiCl 2 (0, 1, 2, 3, 4, 5, 15 M). The obtained rate constants (k 1 ) were subjected to Arrhenius plot, and the E a value was obtained by the equation (2).
G ‡ , H ‡ and S ‡ were obtained by the Eyring equation (3)-(7). Figure S7 summarizes the Arrhenius plots. Table S2 summarizes the kinetic parameters, which are expressed in the bar graph in Figure 5.