CN104262434A - Rna中n6-甲基腺苷的化学脱甲基方法 - Google Patents

Rna中n6-甲基腺苷的化学脱甲基方法 Download PDF

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CN104262434A
CN104262434A CN201410586145.4A CN201410586145A CN104262434A CN 104262434 A CN104262434 A CN 104262434A CN 201410586145 A CN201410586145 A CN 201410586145A CN 104262434 A CN104262434 A CN 104262434A
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methyladenosine
chemical
demethylation
rna
adenosine
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CN104262434B (zh
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周翔
吴晋军
翁小成
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Wuhan University WHU
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    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
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Abstract

本发明涉及用化学方法特异性的使RNA中N6-甲基腺苷脱甲基化，具体方法如下：双氧水和碳酸氢铵在水相中反应生成过氧碳酸HCO4 -，之后N6-甲基腺苷N6位上的N原子作为亲核试剂进攻HCO4 -，经历N6-过氧甲基胞嘧啶（oxm6A），N6-羟甲基胞嘧啶（hm6A）和N6-醛基胞嘧啶（f6A）的中间产物后，最终脱甲基生成腺苷。

Description

RNA中N6-甲基腺苷的化学脱甲基方法
技术领域
本发明涉及RNA中N6-甲基腺苷的化学脱甲基方法，属于化学生物学领域。
背景技术
N6-甲基腺苷是真核生物mRNA中最丰富的化学修饰，也存在于tRNA，rRNA，lnRNA中，自上世纪70年代最初发现以来，目前在哺乳动物植物、苍蝇、减数分裂的酵母和在细胞核增殖的病毒中都发现了m6A的存在。在细胞mRNA中，m6A占所有腺苷的0.1％到0.4％，一条mRNA上大约有3-5个m6A位点，由于N6位的一个甲基不能改变它的Watson-Crick碱基配对的性质，因此不能依靠逆转录的方法来检测m6A，这限制了m6A的功能研究。最近，研究发现FTO和AlkBH5可以作为m6A的脱甲基化酶，证明了m6A的甲基化是一个动态的过程，揭示了哺乳动物中一种新的调节机制，这种可逆的甲基化现象可能影响基因的表达。其次，免疫沉淀法结合二代测序技术为m6A功能的研究提供了可能。这些研究为m6A修饰在生物体内的功能研究提供了的帮助。
发明内容
本发明所要解决的技术问题是在于提供一种有效的化学方法使N6-甲基腺苷脱甲基。
N6-甲基腺苷是上世纪七十年代发现并在最近成为研究的热门，目前已证实了其在生物体内的甲基化是动态的过程，但目前发现的脱甲基化都是酶促反应，并没有有效的化学方法氧化N6-甲基腺苷。据文献报道，碳酸氢铵活化的过氧化氢氧化试剂是简单经济的制备三级胺氮-氧化合物的方法，因此我们想利用此方法氧化N6-甲基腺苷成为硝酮结构，但实际反应后发现，最终产物并不是硝酮结构的氧化物，而是脱甲基产物，在此化学反应中我们又发现并证实了一个新的中间产物N6-过氧甲基腺苷(oxm6A)。反应式如下：
中间产物结构式如下：
本发明的技术方案是：
以N6-甲基腺苷、碳酸氢铵及双氧水为原料，以水作溶剂在37℃下反应4h，HPLC分离得到白色固体，为N6-腺苷。
更具体操作方案是：2mM N6-甲基腺苷与1M碳酸氢铵及200mM双氧水反应，以水作溶剂在37℃下反应24h，HPLC分离得到白色固体，为脱甲基腺苷。
反应机理如下：
双氧水和碳酸氢铵在水相中反应生成过氧碳酸HCO4 -，之后N6-甲基腺苷的N原子作为亲核试剂进攻HCO4 -，经历N6-过氧甲基腺苷，N6-羟甲基腺苷和N6-醛基腺苷的中间产物后，最终脱甲基生成腺苷。
越来越多的证据表明RNA中腺苷的甲基化有着重要的调控作用，N6-甲基腺苷脱甲基化酶的发现证实了m6A动态和可逆的脱甲基化现象，通过控制甲基化酶，脱甲基化酶和RNA底物来控制细胞m6A的动态过程，从而可以在时间和空间上调节细胞活动。我们首次发现非酶促的N6-甲基腺苷脱甲基化反应，并证实其可以在RNA链上进行，在细胞和血清中有着高浓度的碳酸氢根，并有着重要的生物氧化作用，双氧水作为活性氧物质也存在于正常新陈代谢和免疫反应中。
附图说明
图1为N6-甲基腺苷与化合物H2O2和NH4HCO3反应的HPLC图。
图2为从保留时间九到十二分钟的放大图，从图2上可以清楚看到腺苷(A)，N6-羟甲基腺苷(hm6A)，N6-过氧甲基腺苷(oxm6A)和N6-醛基腺苷(f6A)。
具体实施方式
以下以具体实例对本发明的技术方案做进一步的说明。
实施例1：用H2O2和NH4HCO3使N6-甲基腺苷脱甲基化。
取一个1.5mL的PE管，在一个含有200mM H2O2和1M NH4HCO3的反应体系中与2mM的m6A反应，在37℃度下反应24h，得到终产物腺苷(A)及少量中间产物N6-过氧甲基腺苷(oxm6A)。为了证实N-H键和OO-H键的化学位移，我们分别获得了D2O和DMSO-d6中oxm6A的氢谱，在DMSO-d6的溶液中，我们发现了11.71ppm(-N-H)和8.61ppm(-OOH)化学位移的质子，当溶剂换成D2O时，这两个峰消失。说明形成了N6-过氧甲基腺苷的结构。oxm6A：1H NMR(300MHz,DMSO)δ11.71(s,1H),8.61(d,J＝9.0Hz,1H),8.45(s,1H),8.29(s,1H),5.92(d,J＝6.0Hz,1H),5.47(d,J＝6.0Hz,1H),5.31(t,J＝11.1Hz,2H),5.21(d,J＝4.5Hz,1H),4.60(dd,J＝5.4Hz,1H),4.15(d,J＝3.6Hz,1H),3.97(d,J＝3.0Hz,1H),3.68(m,1H),3.54(m,2H).1H NMR(300MHz,D2O)δ8.36(s,1H),8.34(s,1H),6.08(s,1H),5.46(s,2H),4.43(s,1H),4.28(s,1H),3.87(d,J＝13.8Hz,2H).13C NMR(75MHz,DMSO)δ(ppm):155.0,152.8,150.4,141.2,120.6,88.5,86.5,76.0,74.3,71.2,62.3。

Claims (2)

1.一种N6-甲基腺苷的化学脱甲基方法，其特征在于，以N6-甲基腺苷、碳酸氢铵及双氧水为原料，以水作溶剂在37oC下反应4h，HPLC分离得到白色固体，为N6-腺苷。
2.根据权利要求1所述的方法，其特征在于， 2mM N6-甲基腺苷与1M碳酸氢铵及200mM双氧水反应，以水作溶剂在37oC下反应24h，HPLC分离得到白色固体，为脱甲基腺苷。
CN201410586145.4A 2014-10-28 2014-10-28 Rna中n6-甲基腺苷的化学脱甲基方法 Expired - Fee Related CN104262434B (zh)

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CN106967068A (zh) * 2017-05-09 2017-07-21 中国科学院化学研究所 N6‑甲基腺嘌呤的光化学脱甲基方法
CN110093399A (zh) * 2019-04-22 2019-08-06 武汉大学 一种利用二磷酸尿嘧啶脱氧核苷酸检测核酸中腺嘌呤n6位发生甲基化修饰的方法

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CN110093399A (zh) * 2019-04-22 2019-08-06 武汉大学 一种利用二磷酸尿嘧啶脱氧核苷酸检测核酸中腺嘌呤n6位发生甲基化修饰的方法

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