CN112111470A - 一种R-环结合蛋白GST-His6-1/2×HBD及全基因组R-环的检测方法 - Google Patents
一种R-环结合蛋白GST-His6-1/2×HBD及全基因组R-环的检测方法 Download PDFInfo
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Abstract
本发明提供一种R‑环结合蛋白GST‑His6‑1/2×HBD及全基因组R‑环的检测方法，GST‑His6‑1/2×HBD蛋白能够用于结合和定位R‑环，采用以下步骤制备：重组质粒GST‑His6‑1/2×HBD的构建和提取；将重组质粒转入T7Express lysY/Iq感受态细胞，在2×YT培养基中培养并用IPTG诱导蛋白表达；使用Ni‑NTA beads亲和纯化GST‑His6‑1/2×HBD蛋白。借助其对R‑环结构的特异性结合能力，运用靶向剪切及转座酶技术开发出少量细胞，操作简便，信噪比更高的R‑Loop CUT&TAG，以便更好地绘制内源性R‑环基因组图谱，阐释R‑环在基因组中的功能。
Description
技术领域
本发明属于核酸组学领域，具体涉及一种R-环结合蛋白GST-His6-1/2×HBD及全基因组R-环的检测方法。
背景技术
R-环(R-Loop)是RNA侵入双链DNA所形成的一种特殊的三链核酸结构，包括一条DNA:RNA杂合链和一条单链DNA(single strand DNA，ssDNA)。起初R-环被认为只是转录过程形成的副产物，但是近些年的研究发现R-环在细菌、酵母和哺乳细胞中广泛存在，参与调控整个细胞周期，并在基因表达，染色质结构稳定和DNA损伤修复等过程中起着关键作用。与R-环在转录调控和DNA损伤修复的功能相对应，近年来R-环也被报道与许多疾病相关，比如神经性退行性疾病和癌症。例如，在乳腺癌细胞中，肿瘤抑制因子BRCA1或者BRCA2的突变会造成R-环的积累和DNA损伤的加重，这些R-环直接参与肿瘤的发生，同时也是基因组不稳定的标志。在骨髓增生异常综合征(MDS)的研究中，多种剪切因子的突变也会造成造血干细胞R-环水平的紊乱，从而影响基因组稳定性和细胞功能的正常维持。因此，这些证据表明R-环的异常可能是肿瘤发生的驱动因素之一。
然而，因为R-环功能的多样性，在总量上检测R-环水平的变化并不能阐释R-环在上述生理和病理过程中的作用机制。此外，R-环在转录和复制过程中的调控也显示出“亦正亦邪”的特质，因此近十年来许多研究团队一直在开发基因组水平上R-环的精确定位和定量方法。基于染色质免疫共沉淀技术，Chedin等(2012)首次利用识别DNA：RNA杂交体的S9.6单克隆抗体开发了体外富集R-环的高通量测序技术(DNA:RNA immunoprecipitation withdeep sequencing，DRIP-seq)；随后，陈亮等(2015)报道了另外一种基于RNASE H1突变体的R-环检测技术R-CHIP。这些方法虽然将R-环的检测带入了基因组学时代，但是不同检测策略映射出的R-环全基因组图谱存在着很大的差异。此外，这些方法都存在着实验周期长，操作步骤繁琐，信号背景差等缺点，在领域内无法形成一个R-环定位的“金标准”。
发明内容
本发明目的在于提供一种R-环结合蛋白GST-His6-1/2×HBD及全基因组R-环的检测方法，将RNase H1的DNA：RNA杂种结合域HBD进行改良，通过将HBD结构域串联制备特异性识别R-环的重组蛋白GST-His6-1/2×HBD。借助GST-His6-2×HBD对R环结构更强的特异性结合能力，运用最新的靶向剪切及转座酶技术(Cleavage Under Targets andTagmentation，CUT&TAG)进一步开发出少量细胞，操作简便，信噪比更高的R-Loop CUT&TAG，以便更好地绘制内源性R-环基因组图谱，阐释R-环在基因组中的功能。
为实现上述目的，本发明采用以下技术方案：
本发明第一方面提供一种全新的R-环结合蛋白GST-His6-1/2×HBD，所述GST-His6-1/2×HBD蛋白能够用于结合和定位R-环，其采用以下步骤制备：
步骤1：重组质粒GST-His6-1/2×HBD的构建和提取；其中，构建重组质粒GST-His6-1/2×HBD时，质粒结构含有核糖核酸酶RNase H1的DNA：RNA杂交体结合域HBD(HybridBinding Domain)，蛋白纯化标签谷胱甘肽S-转移酶(GST-tag)和组氨酸标签(His-tag)；
步骤2：将重组质粒GST-His6-1/2×HBD转入T7 Express lysY/Iq感受态细胞，在2×YT培养基中培养并用IPTG诱导蛋白表达；
步骤3：使用Ni-NTA beads亲和纯化GST-His6-1/2×HBD蛋白。
进一步地，在步骤2中，诱导蛋白表达使用0.5mM IPTG，诱导时间为5小时，诱导温度为37℃。
进一步地，所述GST-His6-1/2×HBD蛋白对于R-环的结合能力在体外使用凝胶电泳迁移率分析实验(EMSA)检测。纯化后的GST-His6-1/2×HBD蛋白可以与DNA：RNA杂交体在体外孵育，使用凝胶电泳迁移率分析实验检测核酸与蛋白结合导致的阻滞效应，以此来反映纯化的GST-His6-1/2×HBD蛋白活性以及与R-环的结合能力。
进一步地，在细胞水平结合和定位R-环时采用DRIPc-seq方法，其中免疫沉淀R-环的蛋白为GST-His6-2×HBD。
本发明第二方面提供一种全基因组R-环的检测方法(R-Loop CUT&TAG)，采用所述GST-His6-2×HBD蛋白，使用靶向剪切及转座酶技术(CUT&TAG)，特异性纯化细胞内R-环。
进一步地，所述靶向剪切及转座酶技术中，所用的pA-Tn5转座酶组装有适配器接头，接头包括：
Tn5MErev(5'-[phos]CTG TCT CTT ATA CAC ATC T-3')；
Tn5ME-A(5'-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG-3')；
Tn5ME-B(5'-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G-3')。
进一步地，所述靶向剪切及转座酶技术中，R-Loop CUT&TAG测序文库构建之前使用Bst 2.0WarmStart DNA聚合酶完成链置换反应。
进一步地，所述靶向剪切及转座酶技术中，在pA-Tn5转座酶片段化基因组时，反应的缓冲液中添加物包含10％二甲基甲酰胺或者0.85mM ATP，或者两者同时添加。
进一步地，所述靶向剪切及转座酶技术中，可使用单克隆S9.6抗体完成R-LoopCUT&TAG，检测到基因组R-环信号。
进一步地，所述靶向剪切及转座酶技术中在GST-His6-2×HBD蛋白或S9.6抗体孵育过程中，添加RNase A消化后会显著减少富集到的R-环信号。
与现有技术相比，本发明具有以下优点：
(1)本发明使用的GST-His6-1/2×HBD是一种全新的R-环识别因子，解决了之前方法在识别全基因组R-环时由于识别因子特异性问题而产生的争议；
(2)本发明首次提出使用靶向剪切及转座酶技术(CUT&TAG)技术检测全基因组R-环的策略；
(3)R-Loop CUT&TAG耗时短，操作简便，信噪比高，显著提高了R-环检测效率。
附图说明
图1为本发明GST-His6-1/2×HBD重组质粒的构建示意图；
图2为本发明GST-His6-1/2×HBD重组质粒在T7 Express lysY/Iq感受态细胞中，使用原核表达系统设计的蛋白表达与纯化过程；
其中，A图为蛋白表达与纯化流程，B图为蛋白纯化后考马斯亮蓝染色结果；
图3为本发明EMSA实验分析GST-His6-1/2×HBD蛋白对于DNA：RNA杂交体结构的特异性结合能力；
图中，蛋白与核酸结合部分用括号指出，结果显示出GST-His6-1/2×HBD对R-环具有极强的亲和力；
图4为本发明基于已有的DRIPc-seq技术原理，使用GST-His6-2×HBD定位全基因组R-环信号得到的代表性位点示意图；
图中，基因位点示意图显示GST-His6-2×HBD蛋白与S9.6抗体的信号高度一致，阐释了GST-His6-2×HBD蛋白在定位全基因组R-环的可行性；
图5为本发明R-Loop CUT&TAG实验流程示意图；
RNase A可作为对照组添加于抗体结合步骤，以验证该技术在全基因组定位R-环时信号的特异性和真实性；图中，A图表示的是R-Loop CUT&TAG在全基因组定位R-环的实验方法，B图表示的是利用R-环识别因子亲和纯化了基因组R-环后，构建文库用于测序的流程；
图6为本发明R-Loop CUT&TAG在基因组上具有代表性的R-环位点检测到的信号，GST-His6-2×HBD蛋白与S9.6抗体都检测到明显的R-环信号；
图中，在RNase A的消化作用下，R-环的信号显著消失，验证了R-Loop CUT&TAG检测到的R-环的信号特异性和真实性。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。
本发明中“GST-His6-1/2×HBD”指“GST-His6-1×HBD”或“GST-His6-2×HBD”。
【实施例1】重组质粒GST-His6-1/2×HBD的构建
1.引物的设计合成
根据Marcin Nowotny等(2008)提供了不同物种间RNase H1的高度同源性，从addgene上购得含人源的RNase H1编码序列的ppyCAG-RNASEH1-D210N质粒(addgene#111904)，根据质粒信息设计合成特异性引物扩增HBD结构域。在一条引物上加入5个甘氨酸重复序列，表达柔性接头促进串联HBD蛋白表达。引物由上海生工生物工程技术有限公司合成，各引物的序列和编号如下：
(1)HBD基因扩增引物
LP-1：
GCCATATCGAAGGTCGTCATATGATGTTCTATGCCGTGAGGAGGGGC
RP-1：
CTTTGTTAGCAGCCGGTTATCCCCCTCCGCCTCCGCTTGCAGATTTCCT
GACAAAGGC
(2)2×HBD基因扩增引物
LP-2：GTCGTGCATCTGTTGGATATACCATGGGCCATCATCATCAT
RP-2：AGTCAGTCACGATGAATTCCTCCCCCTCCGCCTCC
2.PCR扩增HBD基因与pET16b-His6-HBD重组质粒构建
以ppyCAG-RNASEH1-D210N质粒为模板，使用HBD基因扩增引物与PCR试剂盒(phanta max super-fidelity DNA polymerase，P505-d1，南京诺唯赞生物科技有限公司)特异性扩增HBD基因。用限制性核酸内切酶BamHⅠ和NdeⅠ双酶切pET16b质粒，琼脂糖凝胶电泳后凝胶回收载体片段。利用T5核酸外切酶同源重组修复的原理，将PCR片段与酶切载体以3:1的摩尔比率转化到大肠杆菌DH5α感受态细胞，经过抗性筛选，挑选阳性重组克隆菌种，核苷酸测序验证获得pET16b-His6-HBD重组质粒。
3.GST-His6-1/2×HBD重组质粒构建
以pET16b-His6-HBD重组质粒为模板，使用2XHBD基因扩增引物特异性扩增His6-HBD基因。用限制性核酸内切酶BamHⅠ和EcoRⅠ双酶切pGEX-2T质粒，琼脂糖凝胶电泳后凝胶回收载体片段。采取步骤2同样的同源重组技术，筛选获得GST-His6-1/2×HBD重组质粒，质粒结构示意图见图1。
【实施例2】GST-His6-1/2×HBD蛋白表达与纯化
1.GST-His6-1/2×HBD蛋白表达
将GST-His6-1/2×HBD重组质粒转化到T7 Express lysY/Iq感受态细胞，经过抗性筛选，挑选单克隆菌种培养在含100μg/mL氨苄青霉素钠的2×YT培养基，细菌培养条件设置为37℃，转速200rpm。当细菌的OD600达到0.6时，加入0.5mM IPTG诱导蛋白表达。培养5小时后离心收集细菌颗粒，除去多余的培养基，加入pH7.5的裂解HEX缓冲液(组分包括20mMHEPES，0.8M氯化钠，10％甘油，0.2％Triton X-100，1X蛋白酶抑制剂)。缓冲液与细菌充分混匀后使用细胞破碎仪进行高压破碎，4℃，800psi条件下破碎5分钟，然后高速离心收集上清，此时上清包含有可溶性的GST-His6-1/2×HBD蛋白。
2.GST-His6-1/2×HBD蛋白纯化
依照Ni-NTA Beads(SA004010，常州天地人和生物科技有限公司)说明书亲和纯化GST-His6-1/2×HBD蛋白。在上一步获得的上清中加入1/20体积的Ni-NTA Beads，4℃孵育过夜。在蛋白纯化柱中，依次加入含有20mM咪唑和100mM咪唑的HEX缓冲液，洗去非目的蛋白，直至洗涤液中无蛋白组分洗出，最终使用含250mM咪唑的HEX缓冲液纯化目的蛋白。纯化的目的蛋白在4℃环境中透析过夜，多次透析去除咪唑后，使用超滤管浓缩。使用SDS-PAGE分析GST-His6-1/2×HBD蛋白纯度，然后用BCA蛋白定量试剂盒(PA115-01，北京天根生化科技有限公司)检测蛋白浓度。GST-His6-1/2×HBD蛋白纯化流程与纯化结果见图2。
【实施例3】GST-His6-1/2×HBD蛋白特异性识别R-环结构
(1)凝胶电泳迁移率分析实验(EMSA)检测不同浓度的GST-His6-1/2×HBD蛋白与DNA:RNA杂交体的结合能力，反应条件和体系如下：
用移液器将上述组分混匀，在PCR热循环仪上进行以下反应：25℃孵育30分钟
(2)将步骤(1)的样品加入到6％TBE-PAGE凝胶中进行电泳，电泳缓冲液为0.5×TBE，电泳电压设置为60V，电泳时间为60分钟。
(3)使用GE公司的Typhoon9500扫描凝胶，根据核酸在凝胶内的阻滞现象验证GST-His6-1/2×HBD蛋白能特异性结合DNA:RNA杂交体，具体结果见图3。
【实施例4】基于DRIPc-seq，GST-His6-2×HBD蛋白定位基因组R-环根据Chedin等(2019)设计的DRIPc-seq方案
(1)基因组分离与片段化：消化收集1×107数量的HEK293T细胞，加入20％SDS与蛋白酶K在37℃水浴条件下过夜消化细胞，然后利用酚氯仿法抽提细胞基因组。加入BsrGI，EcoRI，HindIII，SspI和XbaI这五种限制性内切酶后，37℃过夜消化基因组，使用酚氯仿抽提回收片段化的基因组DNA。
(2)R-环分离与纯化：加入GST-His6-2×HBD蛋白在4℃条件下孵育14～17h，利用特异性结合GST标签的磁珠分离纯化HBD蛋白及其结合的R-环结构。经过蛋白酶K消化后，使用酚氯仿抽提获得纯化的R-环。
(3)基于RNA的建库：用DNASE I消化上一步获得的R-环中的DNA，以此得到R-环结构中的RNA链，再通过逆转录反应合成cDNA，经第二链补齐形成双链DNA，然后在DNA两端添加接头标签，建库并在Nova-seq上进行高通量测序。基因组具有代表性的R-环位点见图4。
【实施例5】一种基因组学定位R-环方法：R-Loop CUT&TAG
以HEK293T细胞为例
(1)5×105数量的细胞经过accutase消化后收集，接下来使用PH7.5的洗涤缓冲液(组分包括20mM HEPES，150mM氯化钠，0.5mM亚精胺，1×蛋白酶抑制剂)洗涤2次。细胞中加入10μl活化的刀豆球蛋白A包被磁珠，室温孵育10分钟后洗脱磁珠，完成细胞的固定和穿孔过程。
(2)接下来孵育GST-His6-2×HBD蛋白或者S9.6抗体，以识别细胞内特定的R-环结构。根据相应的蛋白依次孵育anti-GST或者anti-His-tag抗体或者兔抗鼠IgG抗体，以及对应的二抗，实现R-环信号的富集。
(3)使用pH7.5的dig-wash缓冲液(组分包括20mM HEPES，300mM氯化钠，0.5mM亚精胺，1×蛋白酶抑制剂，0.05％毛地黄皂苷)洗涤三次以去除未结合的蛋白。加入带有适配接头的pA-Tn5转座酶复合物，在pH8.5的片段化缓冲液(组分包括10mM TAPS，10mM氯化镁，10％二甲基甲酰胺，0.85mM ATP)中室温孵育1小时完成片段化反应。
(4)然后添加2.25μl 0.5M乙二胺四乙酸,2.75μl 10％SDS和0.5μg蛋白酶K，在55℃条件下反应30分钟终止片段化反应。片段化产物经DNA磁珠回收后使用Bst2.0WarmStart DNA聚合酶完成链置换反应。
(5)最后使用Q5高保真混合体系完成文库构建后用于高通量测序，实验流程设计见图5，检测到的R-环信号见图6。
Claims (10)
1.一种R-环结合蛋白GST-His6-1/2×HBD，其特征在于：所述GST-His6-1/2×HBD蛋白能够用于结合和定位R-环，其采用以下步骤制备：
步骤1：重组质粒GST-His6-1/2×HBD的构建和提取；
其中，构建重组质粒GST-His6-1/2×HBD时，质粒结构含有核糖核酸酶RNase H1的DNA：RNA杂交体结合域HBD(Hybrid Binding Domain)，蛋白纯化标签谷胱甘肽S-转移酶(GST-tag)和组氨酸标签(His-tag)；
步骤2：将步骤1得到的重组质粒GST-His6-1/2×HBD转入T7 Express lysY/Iq感受态细胞，在2×YT培养基中培养并用IPTG诱导蛋白表达；
步骤3：使用Ni-NTA beads亲和纯化步骤2的GST-His6-1/2×HBD蛋白。
2.如权利要求1所述的R-环结合蛋白GST-His6-1/2×HBD，其特征在于：在步骤2中，诱导蛋白表达使用0.5mM IPTG，诱导时间为5小时，诱导温度为37℃。
3.如权利要求1或2所述的R-环结合蛋白GST-His6-1/2×HBD，其特征在于：所述GST-His6-1/2×HBD蛋白对于R-环的结合能力在体外使用凝胶电泳迁移率分析实验(EMSA)检测。
4.如权利要求1所述的R-环结合蛋白GST-His6-1/2×HBD，其特征在于：在细胞水平结合和定位R-环应用DRIPc-seq方法，其中免疫沉淀R-环的蛋白为GST-His6-2×HBD。
5.一种全基因组R-环的检测方法，其特征在于：采用权利要求1所述的GST-His6-2×HBD蛋白，使用靶向剪切及转座酶技术，特异性纯化细胞内R-环。
6.如权利要求5所述的全基因组R-环的检测方法，其特征在于：所述靶向剪切及转座酶技术中，所用的pA-Tn5转座酶组装有适配器接头，接头包括：
Tn5MErev：5'-[phos]CTG TCT CTT ATA CAC ATC T-3'；
Tn5ME-A：5'-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG-3'；
Tn5ME-B：5'-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G-3'。
7.如权利要求5或6所述的全基因组R-环的检测方法，其特征在于：所述靶向剪切及转座酶技术中，R-Loop CUT&TAG测序文库构建之前使用Bst 2.0WarmStart DNA聚合酶完成链置换反应。
8.如权利要求7所述的全基因组R-环的检测方法，其特征在于：所述靶向剪切及转座酶技术中，在pA-Tn5转座酶片段化基因组时，反应的缓冲液中添加物包含终浓度为10％二甲基甲酰胺或者0.85mM ATP，或者两者同时添加。
9.如权利要求8所述的全基因组R-环的检测方法，其特征在于：所述靶向剪切及转座酶技术中，可使用单克隆S9.6抗体完成R-Loop CUT&TAG，检测到基因组R-环信号。
10.如权利要求8或9所述的全基因组R-环的检测方法，其特征在于：所述靶向剪切及转座酶技术中，在GST-His6-2×HBD蛋白或S9.6抗体孵育过程中，添加RNase A消化能够显著减少富集到的R-环信号。
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