T4 RNA Ligase 2 (dsRNA Ligase)

阿拉丁生产的T4 RNA Ligase 2，即T4 RNA连接酶2，是一种ATP依赖的双链RNA连接酶(double-strand RNA ligase, dsRNA Ligase)，也被称为T4 Rnl2 (gp24.1)，可以用于双链RNA进行分子内的环化连接和分子间的线性连接。T4 RNA Ligase 2与T4 RNA Ligase 1 ', "不同的是，对双链RNA中的缺刻(nick)的连接活性要大大高于对单链RNA末端的连接活性。T4 RNA Ligase 2也可用于双链核酸(RNA双链、RNA/DNA杂合链和/或DNA双链)分子内或分子间RNA链的3'羟基和DNA链的5'磷酸基的连接。T4 RNA Ligase 2连接时需要5'磷酸和3'羟基的存在，可以是RNA链或DNA链的5'磷酸基和RNA链的3'羟基之间发生连接反应。T4 RNA Ligase 2催化dsRNA粘端连接的反应过程如下。首先T4 RNA Ligase 2直接消耗ATP，形成中间产物T4 RNA Ligase 2-AMP，并释放焦磷酸；接着，T4 RNA Ligase 2-AMP与dsRNA的缺刻处相结合，并将AMP从中间产物T4 RNA Ligase 2-AMP中转移至dsRNA缺刻处的5'磷酸末端，形成腺苷酰化缺刻dsRNA中间产物；最后，在T4 RNA Ligase 2的催化下，dsRNA缺刻处的3'羟基进攻该缺刻处的5'磷酸基团，形成3'-5'磷酸二酯键，并释放出AMP。本产品酶活性高，连接效率高于国外同类竞争产品。本产品催化双链RNA粘端连接的效果请参考图1。图1. 阿拉丁生产的T4 RNA Ligase 2和N公司的同类产品(Competitor T4 RNA Ligase 2)催化连接dsRNA粘端连接的效果图。 RNA-1：5'-OH-GGGCUUUGCGUGGGUUU-OH-3'；RNA-2 (5'端磷酸化)：5'- pCUAUAGAAACCCACGCAAAGCCC-OH-3'，使用阿拉丁的R0051 Annealing Buffer for RNA oligos (5X)，参考说明书进行退火反应。退火获得的dsRNA，在20µl反应体系中加入图中指定量的本产品或国外N公司的T4 RNA Ligase 2 ，37°C孵育30min进行连接反应，反应完毕后立即取样用15%非变性聚丙烯酰胺凝胶进行电泳分析。如图所示，本产品与N公司的产品相比，实际的连接效率约为N公司产品的2倍，在本反应体系中阿拉丁生产的T4 RNA Ligase 2仅需2U就可以非常充分地完成连接反应。 用途 ： 主要用于连接双链RNA中缺刻的连接(即双链RNA的粘端连接)，也可用于双链结构中，RNA 3'羟基与DNA 5'磷酸基的缺刻连接。 来源 ： 大肠杆菌表达的重组蛋白，表达基因为T4嗜菌体T4 RNA ligase 2。 酶储存溶液 ： 10 mM Tris-HCl (pH7.5), 50 mM KCl, 35 mM (NH4)2SO4, 0.1 mM EDTA, 0.1 mM DTT, 50% (v/v) Glycerol.10X Reaction Buffer：500 mM Tris-HCl (pH 7.5 at 25℃), 20 mM MgCl2, 10 mM DTT, 4 mM ATP. 失活或抑制 ： 85℃加热5min可使T4 RNA Ligase 2失活，或加入蛋白酶K或EDTA可抑制其活性。 注意事项 ： 本产品本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。 为了您的安全和健康，请穿实验服并戴一次性手套操作。 使用说明 ： 1.双链RNA或DNA/RNA杂合双链的退火。将两条单链RNA等摩尔数混合，推荐的终浓度为20µM (10-50µM均可)，90℃孵育1min，然后通过梯度降温至25℃退火形成dsRNA。推荐使用阿拉丁生产的R0051 Annealing Buffer for RNA oligos (5X)，并按照该产品使用说明进行退火反应。DNA和RNA杂合链的退火也可以参考双链RNA的退火条件进行。退火后的双链推荐在-80℃保存。2.对于双链RNA缺刻的连接，参考下表在冰浴中配制如下反应体系：ReagentVolumeFinal ConcentrationDEPC-treated Water16µl-Nicked dsRNA Substrate (20µM)1µl1µM10X T4 Rnl2 Reaction Buffer2µl1XT4 RNA Ligase 2 (10U/µl)1µl0.5U/µlTotal Volume20µl-注意：a.由于涉及RNA操作，需要严格按照RNA操作的规范进行，避免RNase污染，相关试剂和耗材需要经过DEPC处理去除RNase或者确保是RNase free的。本反应体系中涉及双链RNA，双链RNA可以耐受RNase A和T1等。如果涉及单链RNA，包括双链退火后存在部分RNA序列是单链的情况，推荐适量添加R0102 RNase Inhibitor。b.如果同时进行多个连接反应，可以把上表中除Nicked dsRNA Substrate之外的所有溶液和酶提前预混合，然后再分装到各反应管内。c.反应体系中的Nicked dsRNA Substrate的最终浓度可以达到1µM，在该反应体系中可以确保充分连接。实际使用过程中，例如由于底物量有限等原因，可以适当减少Nicked dsRNA Substrate的用量，例如使最终浓度为0.5µM或0.2µM等。3.对于RNA/DNA杂合链中RNA 3'羟基与DNA 5'磷酸基的缺刻连接，参考下表在冰浴中配制如下反应体系：ReagentVolumeFinal ConcentrationDEPC-treated Water10.4µl-Nicked DNA/RNA Substrate (20µM)1µl1µM10X T4 Rnl2 Reaction Buffer2µl1XPEG8000 (50%, RNase Free)4µl0.1MgCl2 (100mM, DEPC-treated)1.6µl8mMT4 RNA Ligase 2 (10U/µl)1µl0.5U/µlTotal Volume20µl-注意：a.由于涉及RNA操作，需要严格按照RNA操作的规范进行，避免RNase污染，相关试剂和耗材需要经过DEPC处理去除RNase或者确保是RNase free的。本反应体系中涉及双链RNA，双链RNA可以耐受RNase A和T1等。如果涉及单链RNA，包括双链退火后存在部分RNA序列是单链的情况，推荐适量添加R0102 RNase Inhibitor。b.如果同时进行多个连接反应，可以把上表中除Nicked DNA/RNA Substrate之外的所有溶液和酶提前预混合，然后再分装到各反应管内。c.反应体系中的Nicked dsRNA Substrate的最终浓度可以达到1µM，在该反应体系中可以确保充分连接。实际使用过程中，例如由于底物量有限等原因，可以适当减少Nicked dsRNA Substrate的用量，例如使最终浓度为0.5µM或0.2µM等。4.连接反应：37℃孵育30min。如果发现效果欠佳可以尝试25℃孵育2h。为了使连接反应更加充分，可以适当延长连接反应时间。5.终止反应：反应结束后加入蛋白酶K或EDTA，即可终止反应。由于T4 RNA Ligase 2热失活需要85℃加热5min，这样可能导致dsRNA或DNA/RNA杂合双链变性，因此通常不建议通过加热方式失活T4 RNA Ligase 2。如果后续无需保持双链状态，则推荐85℃加热5min以失活T4 RNA Ligase 2。 Aladdin's T4 RNA Ligase 2, also known as T4 Rnl2, is an ATP-dependent double-strand RNA ligase (dsRNA Ligase) catalyzing the formation of inter- and intramolecular phosphodiester bonds between the 5'-phosphate terminal and 3'-hydroxyl terminal of dsRNA, RNA/DNA hybrids, and dsDNA. T4 RNA Ligase 2 differs from T4 RNA Ligase 1 in that T4 Rnl2 is significantly more active in joining nicks in dsRNA than ligating ssRNA.T4 RNA Ligase 2 catalyzes the ligation of dsRNA sticky ends as follows. First, T4 RNA Ligase 2 reacts with ATP, producing the intermediate T4 RNA Ligase 2-AMP and releasing pyrophosphate; then T4 RNA Ligase 2-AMP binds to the nick of dsRNA and transfers AMP to the 5' phosphate group of the dsRNA nick to form the adenylated dsRNA intermediate. Finally, catalyzed by T4 RNA Ligase 2, the 3' hydroxyl group at the nick attacks the 5' phosphate group at the same nick, forming a 3'-5' phosphodiester bond and releasing AMP.The enzyme activity of this product is very high, achieving a higher ligation efficiency than other products with similar functions. Please refer to Figure 1 for the performance of this product in ligating the sticky end of dsRNA.Figure 1. Ligation of dsRNA using Aladdin's T4 RNA Rnl2 and T4 Rnl2 from Competitor. RNA-1 ( 5'-OH-GGGCUUUGCGUGGGUUU-OH-3') and RNA-2 (5'- pCUAUAGAAACCCACGCAAAGCCC-OH -3') were annealed in Aladdin's Annealing Buffer for RNA oligos following the manual of this product. As shown in the figure, the ligation efficiency of this product is about 2 times higher than that of the T4 RNA Rnl2 from Competitor.s Application ： Ligation of nicks in ddRNA (i.e., sticky end ligation of dsRNA); ligation of RNA 3'-OH with DNA 5'-P in DNA/RNA hybrids. Source ： Purified from E. coli expressing the T4 RNA ligase 2.Definition of enzyme activity unit: One unit is defined as the amount of enzyme required to ligate 0.4µg of an equimolar mix of 23-mer and 17-mer RNAs in a total reaction volume of 20µl in 30 minutes at 37℃.Enzyme activity assay conditions: 50mM Tris-HCl (pH 7.5), 2mM MgCl2, 1mM DTT, 400µM ATP, incubation at 37℃ for 30 min.Purity: Free of RNase, phosphatase, and DNA endonuclease and exonuclease.Storage buffer: 10mM Tris-HCl (pH 7.5), 50mM KCl, 35mM (NH4)2SO4, 0.1mM EDTA, 0.1mM DTT, 50% (v/v) Glycerol.10X Reaction Buffer: 500mM Tris-HCl (pH 7.5 at 25℃), 20mM MgCl2, 10mM DTT, 4mM ATP. Inactivation or inhibition ： T4 RNA Ligase 2 can be inactivated by heating at 85℃ for 5 min or inhibited by Proteinase K or EDTA. Precautions ： If the ligation substrate is ssRNA, we recommend using T4 RNA Ligase 1 .Additional reagents such as RNase inhibitor and nuclease-free water may be required but not supplied.The T4 Rnl2 Reaction Buffer is suitable for the ligation of nicks in dsRNA. When used for ligating the 3' hydroxyl group of RNA and the 5' phosphate group of DNA in RNA/DNA hybrids, the final concentration of MgCl2 in the reaction should be increased to 10mM and PEG8000 should be added to a final concentratioin of 10-15%, which can significantly increase the activity of T4 RNA Ligation 2 without changing its ligation feature.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation. Instructions for Use ： 1.Annealing of dsRNA or DNA/RNA hybridsMix the two ssRNA strands in equimolar to the recommended final concentration of 20µM (10-50µM), incubate at 90℃ for 1min, and then anneal by gradient cooling to 25℃ to form dsRNA. It is recommend to use ’s Anneaing Buffer for RNA Oligos and follow its instructions for annealing.ssDNA and ssRNA can be annealed to form the DNA/RNA hybrids as described above. After annealing, store the annealed products at -80℃ for future use.2.For the ligation of nicks in dsRNA, set up the following reaction in a nuclease-free microfuge tube on ice:ReagentVolumeFinal ConcentrationDEPC-treated Water16µl-Nicked dsRNA Substrate (20µM)1µl1µM10X T4 Rnl2 Reaction Buffer2µl1XT4 RNA Ligase 2 (10U/µl)1µl0.5U/µlTotal Volume20µl-Note:a.As RNA manipulation is involved, reagents and consumables required should be treated with DEPC to remove RNase. This reaction uses dsRNA which is resistant to RNase A and T1. If ssRNA is involved, we recommend adding RNase Inhibitor (, R0102) in the reaction.b.When multiple reactions are required, prepare a master mix including all reagents except the dsRNA substrate and then dispense to different nuclease-free tubes. Finally, add the dsRNA substrate into each tube.c.The final concentration of Nicked dsRNA Substrate in the reaction can be as high as 1µM to ensure adequate ligation. In case of samples with limited amount of Nicked dsRNA, the final concentration of the Nicked dsRNA Substrate in the reaction can be reduced to 0.5µM or 0.2µM.3.For the ligation of RNA 3'-OH and DNA 5'-P in RNA/DNA hybrids, set up the following reaction on ice:ReagentVolumeFinal ConcentrationDEPC-treated Water10.4µl-Nicked DNA/RNA hybrid (20µM)1µl1µM10X T4 Rnl2 Reaction Buffer2µl1XPEG8000 (50%, RNase Free)4µl10%MgCl2 (100mM, DEPC-treated)1.6µl8mMT4 RNA Ligase 2 (10U/µl)1µl0.5U/µlTotal Volume20µl-Note:a.As RNA manipulation is involved, reagents and consumables required should be treated with DEPC to remove RNase. This reaction uses dsRNA which is resistant to RNase A and T1. If single-stranded RNA is involved, we recommend adding RNase Inhibitor in the reaction.b.When multiple reactions are required, prepare a master mix including all reagents except the DNA/RNA hybrid and then dispense to different nuclease-free tubes. Finally, add the DNA/RNA hybrids into each tube.c.The final concentration of nicked DNA/RNA hybrid in the reaction can be as high as 1µM to ensure adequate ligation. In case of samples with limited amount of nicked DNA/RNA hybrids, the final concentration of the nicked DNA/RNA hybrid in the reaction can be reduced to 0.5µM or 0.2µM. 4.Incubate at 37℃ for 30min. If the ligation efficiency is low, incubate at 25℃ for 2 hours. The incubation time can be extended approximately to make the ligation more adequate.5.Terminate the reaction by adding proteinase K or EDTA.Heat inactivation of T4 RNA Ligase 2 requires heating at 85℃ for 5 min, which may result in denaturation of dsRNA or DNA/RNA hybrid strands, so heat inactivation is not usually recommended. However, if the duplex state is not required subsequently, the reaction can be terminated by heating at 85℃ for 5 min.

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