Modified and CpG sequence unit contained oligodeoxynucleotide molecule and application thereof
A deoxynucleotide and oligomerization technology, applied in biochemical equipment and methods, antineoplastic drugs, allergic diseases, etc., can solve the problem of low bioavailability, achieve good oral bioavailability, improve immune response, Good immune regulation effect
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Embodiment 1
[0033] Embodiment 1, the influence of azide and alkynyl concentration, catalyst, reaction time on CuAAC reaction
[0034] A dipeptide AF is selected to optimize the reaction conditions, and the optimized method can be extended to the click reaction between other small molecules and CpG ODN. The premise of all changes in reaction conditions is that the reaction is carried out under shaking at room temperature.
[0035] Table 1 Experimental design with different concentrations of azide and alkynyl groups, different proportions of catalysts and different reaction times
[0036]
[0037] figure 1 It is the PAGE diagram of the click reaction under different reaction conditions. In (a) and (b), M represents a 20bp DNA marker, and lane 1 is an unmodified CpG ODN; perform PAGE electrophoresis according to the design in Table 1; figure 1 The reaction conditions in (a) refer to the conditions of the classic small molecule click reaction [Rostovtsev, V.V., et al., A stepwise hui...
Embodiment 2
[0045] Example 2, the ratio of azide small molecule to CpG ODN and the influence of reaction buffer on CuAAC reaction
[0046] After determining the optimal reaction time, clarifying the role of TBTA, optimizing the concentration of CpG ODN and AF and the ratio of the catalyst, the effect of different ratios of AF and CpG ODN and the reaction buffer on the click reaction was investigated, and the coupling product was determined purification method.
[0047] Table 2 Experimental design of different buffers, different ratios of AF and CpG ODN
[0048]
[0049] figure 2 In (a) and (b), M represents 20bp DNA marker, lane 1 is unmodified CpG ODN; (a) according to Table 2 Designed for electrophoresis; (b) PAGE verification of the purification method, lane 3 is purified CpG-AF;
[0050] figure 2 In (a), 2M pH 7.0 triethylamine acetate buffer was added to the reaction system of lane 2 and lane 3, and there was no buffer in the reaction system of lane 4 and lane 5; the react...
Embodiment 3
[0052] Example 3, the above CuAAC reaction conditions are applied to other small molecules
[0053] Take the fatty acid lauric acid as an example, image 3 Schematic diagram of the CuAAC reaction of azidolauric acid and alkyne-modified CpG ODN;
[0054] Reactant stock solution configuration;
[0055] ①0.5mM Alkyne-modified CpG ODN stock solution:
[0056]Centrifuge the centrifuge tube containing the alkyne-modified CpG ODN powder first. Each tube contains 25 nmol of CpG ODN, add 50 μl of ultrapure water, shake to mix evenly, and store at -20°C.
[0057] ②0.02M azidolauric acid stock solution: 2.3mg azidolauric acid, add 353μl DMF, shake to dissolve, seal and store at 4°C.
[0058] ③ 4mM TBTA stock solution: Weigh 4.2mg TBTA powder, dissolve in 2ml DMF, shake to mix evenly, and store at -20°C.
[0059] ④0.5mM CuSO 4 Stock solution: weigh 12.5mg CuSO 4 ·5H 2 O, dissolved in 1ml ultra-pure water, ready to use now.
[0060] ⑤ 0.2M Sodium Ascorbate stock solution: Weigh ...
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