Summary

测量小核苷酸基质的多核苷酸磷酸化的非放射性测定

Published: May 08, 2020
doi:

Summary

该协议描述了一种非放射性检测,用于测量小DNA和RNA基质上多核苷酸激酶(PNKs)的激酶活性。

Abstract

多核苷酸激酶(PNKs)是催化DNA和RNA寡核苷酸5’羟基端磷酸化的酶。可以直接或间接地对PNK的活动进行量化。此处介绍的是一种直接的体外测量 PNK 活性的方法,它依赖于荧光标记的寡核苷酸基质和聚丙烯酰胺凝胶电泳。这种方法提供磷酸化产品的分辨率,同时避免使用放射性标记基板。协议详细介绍了如何设置磷酸化反应,准备和运行大型聚丙烯酰胺凝胶,并量化反应产品。这种测定技术上最具挑战性的部分是浇注和运行大型聚丙烯酰胺凝胶;因此,提供了克服共同困难的重要细节。该协议针对Grc3进行了优化,Grk是一种PNK,它结合其结合伙伴Las1核酸酶组装成一个义务性前核糖体RNA处理复合物。然而,此协议可以适应测量其他PNK酶的活性。此外,这种测定也可以被修改,以确定反应的不同成分的影响,如核苷三磷酸盐,金属离子和寡核苷酸。

Introduction

多核苷酸激酶(PNK)在许多DNA和RNA处理途径中起着至关重要的作用,如DNA修复和核糖体组装1、2、3、4、5。,2,3,4,5这些基本酶催化终端(伽马)单磷酸盐从核苷三磷酸盐(NTP,最常见的ATP)转移到核苷酸基质的5’羟基端。PNK最具有最良好特征之一是细菌T4,PNK,它具有广泛的基质特异性,被分子生物学实验室大量利用,将放射性同位素标签整合到DNA或RNA基质,,,6、7、8、9、10、11、127,85个术语91011PNK酶的另一个例子是CLP1,它存在于尤卡里亚、尤菌和阿奇亚,并牵连到几个RNA处理途径4,13,14,15。,14,154,

从历史上看,大多数测量多核苷酸激酶活性的测定都依赖于放射性同位素标记和随后的自体造影,5,16。近年来,还开发了一些用于测量PNK活性的附加检测方法,包括单分子方法、微芯片电泳、分子信标,以及基于,色度和发光的测定17、18、19、20、21、22。,18,19,2021,22虽然许多新方法提供了增强的检测限值并避免使用放射性,但每种方法都有缺点,例如成本、对固定树脂的依赖以及基板选择的限制。

Grc3是一种多核苷酸激酶,在核糖体前RNA2、3、23,3,的处理中起着关键作用。Grc3与内白蛋白酶Las1形成一个义务复合体,它切割了前核糖体RNA3的内部转录空间2(ITS2)。Las1的 ITS2 的裂解产生一种含有 5′ 羟基的产品,随后由 Grc3 激酶3 磷酸化。为了研究Grc3的核苷酸和基质特异性,需要一种廉价的测定方法,允许对不同的寡核苷酸基质进行检测。因此,利用荧光标记基材开发了PNK磷酸化测定。该测定成功地用于确定Grc3可以利用任何NTP进行磷酸转移活性,但有利于ATP24。该协议调整原始测定,以测量Grc3的PNK活性,其RNA模拟其前核糖体RNA基质(SC-ITS2,表1)。这种基于荧光的方法的一个具有挑战性的方面是依靠大型聚丙烯酰胺凝胶来有效解决磷酸化和非磷化基材。该协议提供了有关如何倒这些大凝胶的具体细节,并避免在这样做时常见的陷阱。

使用RNA需要特别小心,因为它极易降解。有简单的预防措施,可以采取,以限制核糖核酸酶污染。单独的RNA工作站,可以很容易地用含有RNase抑制剂的清洁剂处理,通常是有帮助的。处理样品时始终佩戴手套,并且必须使用无 RNase 认证的耗材。由于水是另一种常见的污染源,因此最好使用新鲜纯净的水,并使用 0.22 μm 过滤器对所有溶液进行消毒。

Protocol

1. 准备 准备缓冲液和试剂。 通过组合 20 μL 的 1 M Tris (pH = 8.0)、40 μL 的 5 M 氯化钠、2.5 μL 的 2 M 氯化镁、100 μL 的 50%(v/v)甘油和无 1 mL 的 RNase 水,制成 1x 反应缓冲液,达到 1 mL 的总体积。 通过组合 4.8 g 尿素、200 μL 的 1 M Tris (pH = 8.0)、20 μL 的 0.5 M EDTA (pH = 8.0)、0.5 mL 的 1% (w/v) 肉酚蓝色和无 Rse-0 水,使尿素加载染料达到 10 mL 的总体积。 通过组合 108 g …

Representative Results

图1中显示了ATP滴定与固定数量的Las1-Grc3复合物的成功代表性 变性凝胶。酶的加入导致SC-ITS2 RNA基质的Las1中导RNA裂解,导致定义的RNA片段(5-OH C2 RNA)。添加ATP后,C2 RNA片段由Grc3 PNK(5-P C2 RNA)磷酸化。在变性凝胶中,磷酸化RNA的迁移速度比其未磷化RNA的迁移速度要快。如图 2所示,C2RNA片段的磷酸化可以通过绘制未磷化和磷酸?…

Discussion

所述是测量Grc3 PNK在荧光标记核苷酸基质基质上激酶活性的测定方法。该协议可以通过调整反应缓冲液和寡核苷酸基质来应用于其他PNK酶的特性。例如,协议要求跟踪量为 EDTA。添加EDTA是有益的,原因有二:首先,这种方法有利于镁结合Grc3,防止酶与混合物中微量的污染金属结合。其次,少量的EDTA抑制污染金属依赖性核糖核酸的活动,而不会破坏相关的Las1金属独立核糖核糖的活性。EDTA 的浓度可…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢安德鲁·西克马博士和安德里亚·卡明斯基对这份手稿的批判性解读。这项工作得到了美国国家卫生研究院校内研究项目的支持;美国国家环境卫生科学研究所(NIEHS;ZIA ES103247 至 R.E.S) 和加拿大卫生研究所 (CIHR; 146626 到 M.C.P)。

Materials

0.4 mm 34-well comb BioRad 1653848
0.4 mm spacer BioRad 1653812
0.5 M EDTA ph 8.0 KD Medical RGF-3130
1M Magnesium Chloride KD Medical CAC-5290
1M Tris pH 8.0 KD Medical RGF-3360
40% Acrylamide/Bis Solution 29:1 BioRad 1610146
5M Sodium Chloride KD Medical RGF-3720
ammonium persulfate (APS) BioRad 161-0700
ATP Sigma A2383-1G
boric acid Sigma B0394
bromophenol blue sodium salt Sigma B5525-5G
Glass Plates Thomas Scientific 1188K51
Hoefer SQ3 Sequencer Hoefer N/A
Image J Software N/A N/A https://imagej.nih.gov/ij/
Labeled RNA oligonucleotides IDT Custom Order
Pharmacia EPS 3500 Power Supply Pharmacia N/A
Steriflip 0. 22 um Filter Millipore 5FCP00525
TEMED BioRad 161-0800
tris base Sigma TRIS-RO
Typhoon FLA 9500 gel imager GE Healthcare N/A
Ultra Pure DEPC Water Invitrogen 750023
Ultra Pure Glycerol Invitrogen 19E1056865
urea Fisher Chemical U15-500

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Cite This Article
Pillon, M. C., Stanley, R. E. Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates. J. Vis. Exp. (159), e61258, doi:10.3791/61258 (2020).

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