WO2019169701A1 - 一种用SLFN13从总RNA中纯化总mRNA的方法 - Google Patents

一种用SLFN13从总RNA中纯化总mRNA的方法 Download PDF

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WO2019169701A1
WO2019169701A1 PCT/CN2018/083874 CN2018083874W WO2019169701A1 WO 2019169701 A1 WO2019169701 A1 WO 2019169701A1 CN 2018083874 W CN2018083874 W CN 2018083874W WO 2019169701 A1 WO2019169701 A1 WO 2019169701A1
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slfn13
total rna
mrna
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rna
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高嵩
杨金玉
谢伟
邓翔宇
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中山大学肿瘤防治中心
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Priority to US16/625,486 priority Critical patent/US20210071164A1/en
Priority to EP18908351.2A priority patent/EP3636756B1/en
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    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease
    • C12Q2521/301—Endonuclease

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  • the present invention belongs to the field of biotechnology, and in particular relates to a method for purifying total mRNA from total RNA using SLFN13 (Schlafen 13).
  • mRNA is a necessary stage of gene expression, which can reflect the transcription and expression information of a specific cell or tissue under a certain functional state, and is closely related to the characteristics and growth state of the cell.
  • High-throughput sequencing of transcriptome mRNA is currently a highly popular method for obtaining complete sequence information of RNA with poly-A tail in one test, analyzing gene expression, cSNP, brand new transcription, and new isomerism.
  • Comprehensive transcriptome information such as body, splice sites, allele-specific expression and rare transcription.
  • the first important step in the sequencing experiment is to extract the total RNA of the target cell or tissue and obtain high-quality total mRNA with good integrity and high purity as much as possible, and then to establish a detailed cDNA library through reverse transcription process.
  • rRNA accounts for 75%-85% of total RNA extracted from organisms
  • tRNA accounts for 10%-16%
  • mRNA only accounts for 1%-5%
  • high quality purification of mRNA from total RNA while ensuring its integrity is a relatively difficult and critical step in the process of building a library.
  • mRNA there are mainly two kinds of methods to achieve relative purification of mRNA: one is to use the characteristics of poly dT at the end of mRNA, design a matrix that specifically binds to poly dT, and specifically separate mRNA from total RNA; the other is design A substrate that specifically binds to a conserved region of rRNA, the highest content of rRNA in total RNA can be isolated from total RNA as much as possible to obtain relatively purified mRNA.
  • RNA degradation can seriously affect the effect of building a database, which not only leads to the loss of important information, but also introduces many errors and errors. More importantly, the purification effects of the two methods are not very satisfactory: in the first method, the purified mRNA can only guarantee 40-70% integrity, which will affect the accuracy of the sequencing data and the data set. The display of the difference between the two.
  • the second method also has an effect on the integrity of the mRNA, although the effect is smaller than the first method, but the mRNA obtained is inferior to the first method, because although this method can remove most of the rRNA, the total RNA The higher content of tRNA cannot be removed, and the final total mRNA obtained is still less than 30% pure.
  • transcriptome sequencing With the development and advancement of sequencing technology, more and more researchers tend to use transcriptome sequencing to obtain key data for related topics. In order to obtain accurate and reliable data by sequencing the transcriptome, it is critical to obtain high-quality total mRNA from total RNA and must be strictly controlled. Because the quality of total mRNA directly determines the effectiveness of subsequent database construction and the validity of sequencing data, if the quality and integrity of total mRNA are not up to standard, there may be large errors and distortions in subsequent sequencing analysis results.
  • the object of the present invention is to overcome the defects and shortcomings of the above-mentioned prior art methods for purifying total mRNA obtained from total RNA (the steps are cumbersome, the purification effect is not satisfactory, and the quality and integrity of mRNA are difficult to ensure), etc.
  • SLFN13 is a method for purifying total mRNA from total RNA.
  • the invention utilizes the specific digestion of tRNA and rRNA by SLFN13 to purify total mRNA from total RNA, which not only greatly improves the purity of total mRNA, but also simplifies the experimental procedure, saves time and ensures the stability and integrity of total mRNA, thereby ensuring Accuracy and effectiveness of subsequent library sequencing data or other relevant experimental analysis.
  • the object of the present invention is achieved by the following technical scheme: a method for purifying total mRNA from total RNA using SLFN13, and the specific steps are as follows:
  • Total RNA extraction Total RNA was extracted from the sample by the traditional TRIzol-chloroform method
  • the enzyme digestion system ie, the enzyme is digested with 5 pmol of SLFN13 per ug of total RNA, providing an enzyme concentration of 50 uM
  • the composition of the 10 ⁇ digestion buffer is: 400 mM Tris-HCl (pH 8.0), 200 mM KCl, 40 mM MgCl 2 and 20 mM DTT; incubation at room temperature for 30 min;
  • Figure 2-5 shows that SLFN13-N can specifically cleave tRNA and rRNA in total RNA, and digest it into fragments within 100 nt; If there are differences in composition, the amount of enzyme and the time of enzyme digestion may be appropriately increased or decreased, and it is recommended to fluctuate within 30%;
  • SLFN13-N can be inactivated by heating at 70 ° C for 15 minutes to obtain purified total mRNA.
  • the sample described in the step (1) is one of a cell sample or a tissue sample.
  • total RNA can be extracted by other effective methods to ensure the total RNA quality and integrity.
  • the SLFN13 described in the step (2) is one of the SLFN13 full length or the N-terminal domain of SLFN13.
  • the N-terminal domain of SLFN13 (collectively referred to as SLFN13-N) is the amino acid sequence 1-355 of human SLFN13 (hSLFN13-N, Gene ID: 146857) or the amino acid sequence of rat SLFN13 1-353 (rSLFN13-N, Gene ID: 303378).
  • the human SLFN13 has a Gene ID of 146857 and is mainly purified using 1-355 of its amino acid sequence.
  • the rat SLFN13 has a Gene ID of 303378 and is mainly purified using 1-353 of its amino acid sequence.
  • the N-terminal domain of the SLFN13 can be prepared by the following expression purification method:
  • SLFN13 The N-terminal domain of SLFN13 (amino acid sequence 1-355 of human SLFN13, hSLFN13-N, Gene ID: 146857 and amino acid sequence of rat SLFN13 1-353, rSLFN13-N, Gene ID: 303378; collectively referred to as SLFN13- N) separately constructed into pET28 vector, after sequencing and correct, transform the plasmid into Rossetta (DE3) expression strain; pick monoclonal into 100ml LB medium supplemented with kanamycin and ampicillin double pre-culture, 12 After 16 hr, the bacterial solution was transferred to 5 L of TB medium supplemented with double antibody at a ratio of 1:100 to expand the culture at 37 ° C.
  • SLFN13- N amino acid sequence 1-355 of human SLFN13, hSLFN13-N, Gene ID: 146857 and amino acid sequence of rat SLFN13 1-353, rSLFN13-N, Gene ID: 303378; collective
  • the digested tRNA and rRNA fragments are removed in combination with the corresponding small RNA purification kit to obtain high purity.
  • Total mRNA is obtained after the enzyme is inactivated in step (3).
  • the total mRNA obtained by the preparation of the invention can be directly used in subsequent database construction experiments.
  • the invention breaks the traditional concept of RNA purification, and introduces a specific RNA endonuclease to remove the RNA molecules to be removed from the total RNA, which is simple and efficient.
  • the advantages of this patent can be summarized as follows:
  • the purification process of the invention does not require the use of too much additional RNA purification medium, such as a specific RNA binding matrix, a special RNA purification buffer, and the like.
  • the most critical step is purification to obtain an active endonuclease which can be expressed by an ordinary E. coli strain and separated by a nickel matrix affinity chromatography in combination with molecular sieves to obtain a protein having a purity of more than 90%.
  • 3L inducible expression strain can be cultured, about 50mg protein (about 12mM) can be obtained after purification, and the enzymatic cleavage efficiency of the enzyme is very high in vitro, and 4pmol enzyme can cleave 1ug RNA substrate at room temperature, it takes only 10-20 minutes. . Therefore, the time to purify the enzyme is short, the cost is low, but it can be used multiple times.
  • RNA purification matrix was omitted, and the processes of matrix balance, RNA specific binding and elution were skipped. Simply add the appropriate amount of enzyme to the RNA digestion system in one step. The enzyme treatment stage only needs to be left to stand or simply rotate. No additional human monitoring is required. Subsequent RNA fragments can be skipped through the purification step because of the small fragment of RNA. Fragments do not interfere too much with building a larger molecular weight mRNA. The whole process is easy to master, and it is not easy to introduce errors, even the novice can quickly and skillfully operate.
  • the purification effect is good, and the tRNA and rRNA can be selectively removed in one step to ensure the purity and integrity of the total mRNA.
  • the invention mainly relies on a specific endonuclease to digest the RNA component to be excluded, and in view of the restriction enzyme specificity and specificity, the tRNA and rRNA enzymes which are the most abundant in total RNA can be used at one time. Cut and remove, more thoroughly than other purification methods. Since the enzyme has no enzymatic activity on single-stranded RNA, mRNA integrity can be ensured as much as possible.
  • RNA is easily degraded in the air.
  • the probability of degradation of the mRNA introduced during the experimental operation is greatly reduced, and the quality of the total mRNA obtained by the purification is ensured.
  • Fig. 1 is a graph showing the results of SDS-PAGE analysis of samples prepared from the collection tubes corresponding to the elution peaks of the monomer proteins after purification of hSLFN13-N and rSLFN13-N.
  • Figure 2 shows the results of urea gel electrophoresis analysis after selective digestion of tRNA by SLFN13-N.
  • Figure 3 shows the determination of the restriction enzyme site of SLFN13-N and its detection of the maturation activity of mature tRNA in vivo.
  • Figure a and Figure b show the enzymes of small RNA extracted from 293T cells by hSLFN13 and rSLFN13 and their related mutations, respectively. Cut the effect.
  • Figure c and Figure d show the enzymatic cleavage effect of hSLFN13 and rSLFN13 and their related mutations on the small RNA extracted from HeLa cells.
  • Small RNA mainly contains tRNA and 5S and 5.8S rRNA.
  • Figure 4 Northern blot method to verify the detection of the digestive activity of tRNA and rRNA in total RNA extracted by SLFN13-N.
  • Panels a-c detect the cleavage of these three mature tRNAs by SLFN13 using probes specific for tRNASer, tRNAGly and tRNALys, respectively.
  • Figure d is a cleaving of 5S rRNA by SLFN13 using a 5S rRNA probe.
  • Figure 5 Enzyme digestion results of rRNA in total RNA extracted by SLFN13-N.
  • Panel a shows the enzymatic cleavage of total RNA extracted from 293T cells by SLFN13 (hSLFN13 and rSLFN13) and their associated mutations.
  • Figure b shows the enzymatic cleavage of total RNA extracted from HeLa cells by SLFN13 (hSLFN13 and rSLFN13) and their related mutations.
  • SLFN13 The N-terminal domain of SLFN13 (amino acid sequence 1-355 of human SLFN13, hSLFN13-N, Gene ID: 146857 and amino acid sequence of rat SLFN13 1-353, rSLFN13-N, Gene ID: 303378; collectively referred to as SLFN13- N) separately constructed into pET28 vector, after sequencing and correct, transform the plasmid into Rossetta (DE3) expression strain; pick monoclonal into 100ml LB medium supplemented with kanamycin and ampicillin double pre-culture, 12 After 16 hr, the bacterial solution was transferred to 5 L of TB medium supplemented with double antibody at a ratio of 1:100 to expand the culture at 37 ° C.
  • SLFN13- N amino acid sequence 1-355 of human SLFN13, hSLFN13-N, Gene ID: 146857 and amino acid sequence of rat SLFN13 1-353, rSLFN13-N, Gene ID: 303378; collective
  • the present invention provides a method for purifying total mRNA from total RNA using SLFN13, and the specific steps are as follows:
  • Total RNA extraction Cell or tissue samples can be extracted from intact total RNA using the traditional TRIzol-chloroform method (if the sample is specific, other applicable methods can be considered to ensure the total RNA quality and integrity).
  • RNA For example, 83% and 3% of the total RNA, 10ul concentration of 1ug / ul total RNA, add 1ul 50uM SLFN13, add 2ul 10 ⁇ digestion buffer, add 7ul ddH2O with 20ul enzyme digestion system (ie per ug Total RNA was digested with 5 pmol of SLFN13 to provide an enzyme concentration of 50 uM); 10 ⁇ digestion buffer was composed of 400 mM Tris-HCl (pH 8.0), 200 mM KCl, 40 mM MgCl 2 and 20 mM DTT; That is to say; Figure 2-5 shows that SLFN13-N can specifically cleave tRNA and rRNA in total RNA until it is cut into pieces within 100 nt. If there is a difference in the composition of the total RNA source, the amount of the enzyme may be appropriately increased or decreased, and it is recommended to fluctuate within 30%.
  • the SLFN13-N can be inactivated by heating at 70 ° C for 15 minutes;
  • step (3) If the purity of the mRNA is more stringent, after the step (3), the digested tRNA and rRNA fragments are removed in combination with the corresponding small RNA purification kit to obtain high-purity mRNA and then constructed. step.
  • Figure 2 is a selective digestion of tRNA by SLFN13-N.
  • SLFN13-N was digested with different types of nucleic acid substrates in vitro for 30 minutes, and analyzed by urea gel electrophoresis. The results showed that SLFN13-N only specifically cleaves tRNA.
  • Figure 3 Determination of the restriction site of SLFN13-N and its restriction enzyme activity against mature tRNA in vivo.
  • Figure 4 Northern blot method to verify the enzymatic activity of SLFN13-N on tRNA and rRNA in total RNA extracted from vivo.
  • the probes for tRNASer, tRNAGly, tRNALys and 5S rRNA were designed and labeled with P32 at the 5' end.
  • SLFN13-N was incubated with the total RNA extracted from the cells, and then separated and transferred to the membrane. The needle is hybridized with the digested product.
  • the results showed that SLFN13-N had significant enzymatic cleavage activity on tRNA and 5S rRNA in total RNA extracted from cells. As time and enzyme amount increased, 5S rRNA could be digested into fragments.
  • Figure 5 The enzymatic cleavage activity of SLFN13-N on rRNA in total RNA extracted from in vivo.
  • SLFN13-N and its related enzymes were incubated with total RNA extracted from HEK-293T cells and HeLa cells, respectively.
  • the enzymatic cleavage effect was significantly enhanced with increasing enzyme concentration, and rRNA could be gradually fragmented with time.
  • the invention breaks the traditional concept of RNA purification, and introduces a specific RNA endonuclease to remove tRNA and rRNA molecules from total RNA, which is simple and efficient.
  • the advantages of this patent can be summarized as follows:
  • the purification process of the invention does not require the use of too much additional RNA purification medium, such as a specific RNA binding matrix, a special RNA purification buffer, and the like.
  • the most critical step is purification to obtain an active endonuclease which can be expressed by an ordinary E. coli strain and separated by a nickel matrix affinity chromatography in combination with molecular sieves to obtain a protein having a purity of more than 90%.
  • 3L inducible expression strain can be cultured, about 50mg protein (about 12mM) can be obtained after purification, and the enzymatic cleavage efficiency of the enzyme is very high in vitro, and 4pmol enzyme can cleave 1ug RNA substrate at room temperature, it takes only 10-20 minutes. . Therefore, the time to purify the enzyme is short, the cost is low, but it can be used multiple times.
  • the purification effect is good, and the tRNA and rRNA can be selectively removed in one step to ensure the purity and integrity of the mRNA.
  • the invention mainly relies on a specific endonuclease to digest the RNA component to be excluded, and in view of the restriction enzyme specificity and specificity, the tRNA and rRNA enzymes which are the most abundant in total RNA can be used at one time. Cut and remove, more thoroughly than other purification methods. Since the enzyme has no enzymatic activity on single-stranded RNA, mRNA integrity can be ensured as much as possible.
  • RNA is easily degraded in the air.
  • the probability of degradation of the mRNA introduced during the experimental operation is greatly reduced, and the quality of the total mRNA obtained by the purification is ensured.

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Abstract

提供了一种用SLFN13从总RNA中纯化总mRNA的方法，具体步骤如下：(1)总RNA提取；(2)利用SLFN13对总RNA中的tRNA与rRNA进行酶切；(3)酶切结束后可以直接于70℃加热15分钟将酶灭活，获得纯化的总mRNA。

Description

一种用SLFN13从总RNA中纯化总mRNA的方法 技术领域
本发明属于生物技术领域，特别涉及一种用SLFN13(Schlafen13)从总RNA中纯化总mRNA的方法。
背景技术
mRNA是基因表达的必经阶段，可以体现特定细胞或组织在某一功能状态下的转录情况和表达信息，与细胞的特性和生长状态等密切相关。转录组mRNA高通量测序是目前备受青睐的高效方法，仅需一次试验即可快速获得带poly-A尾巴的RNA的完整序列信息，可分析基因表达、cSNP、全新的转录、全新异构体、剪接位点、等位基因特异性表达和罕见转录等全面的转录组信息。测序实验过程中第一个重要步骤是提取目的细胞或组织的总RNA并尽可能的得到完整性好、纯度高的高质量总mRNA，然后才能更有效的经逆转录过程建立详尽的cDNA文库，最终得到准确可靠的测序结果。然而，从生物体中提取得到的总RNA中，rRNA占75％～85％，tRNA占10％～16％，而mRNA仅占1％～5％，且mRNA分子种类繁多，分子量大小不均一，表达丰度也有差异。因而将mRNA从总RNA中高质量的纯化出来同时保证其完整性是建库过程中相对困难又非常关键的步骤。目前主要有两类方法来实现mRNA的相对纯化：一类是利用mRNA的末端含有poly dT的特性，设计特异结合poly dT的基质，从总RNA中将mRNA特异的分离出来；另一类是设计可以与rRNA保守区段特异性结合的基质，将总RNA中含量最高的rRNA尽可能的从总RNA中分离出去，得到相对纯化的mRNA。
由于RNA具有相对不稳定的性质，在体外尤其是暴露在空气中容易降解，以上两类纯化方法的过程相对繁琐，不容易在短时间内完成，大大增加了RNA的降解风险。RNA降解会严重影响建库的效果，不仅会导致重要信息的缺失，还可能引入很多错误和误差。更重要的是，这两种方法的纯化效果均不十分理想：第一类方法中，纯化得到的mRNA仅能保证40-70％的完整性，这会影响测序数据的准确性和数据组之间差异的显示。第二类方法同样对mRNA的完整性有影响，虽然影响较第一类方法小，但其得到的mRNA纯度较第一类方法差，因为尽管该类方法可以去除绝大部分rRNA，但总RNA中含量较高的tRNA无法去除，最终得到的总mRNA的纯度仍在30％以下。
随着测序技术的发展和进步，越来越多研究者倾向于利用转录组测序的方式来获得相关课题的关键数据。要想通过转录组建库测序的方式得到准确可靠的数据，从总RNA中获得高质量的总mRNA这一环节是非常关键且必须严格控制的。因为总mRNA的质量直接决定了后续建库的效果及测序数据的有效性，若总mRNA的质量和完整性不合标准，后续得到的测序分析结果中可能会存在较大误差而失真。
发明内容
本发明的目的在于克服上述现有技术中从总RNA中纯化得到的总mRNA的方法的缺陷与不足(步骤繁琐、纯化效果不理想及mRNA的质量和完整性难以保证等)，提供一种用SLFN13从总RNA中纯化总mRNA的方法。本发明利用SLFN13对tRNA和rRNA的特异性酶切从总RNA中纯化总mRNA，不仅大大提高总mRNA的纯度，还能简化实验流程、节约时间并并保证总mRNA稳定性和完整性，从而保证后续建库测序数据或其它相关实验分析的准确性和有效性。
根据以上两类较为传统的纯化方式的缺点，我们以一种新颖的方式实现总mRNA的纯化，引入特异性针对tRNA和rRNA的内切酶，将总RNA中含量最高的两类RNA酶切至小分子碎片而不影响mRNA的完整性，此方法简单便捷，不需要经过纯化基质的结合和洗脱等繁琐过程大大降低了mRNA的降解几率，又能保证mRNA的纯度。
本发明的目的通过下述技术方案实现：一种用SLFN13从总RNA中纯化总mRNA的方法，具体步骤如下：
(1)总RNA提取：利用传统的TRIzol-氯仿方法从样品中提取完整的总RNA；
(2)tRNA与rRNA的酶切：以纯化10ug总RNA为例，取10ul浓度为1ug/ul的总RNA，加入1ul 50uM的SLFN13，加2ul 10×酶切缓冲液，加7ul ddH2O配成20ul的酶切体系(即每ug总RNA用5pmol SLFN13进行酶切，提供的酶浓度为50uM)；10×酶切缓冲液的成分为：400mM Tris-HCl(pH 8.0),200mM KCl,40mM MgCl2和20mM DTT；室温孵育酶切30min即可；图2-图5已经证明SLFN13-N可以特异性的酶切总RNA中的tRNA和rRNA，将其酶切为100nt以内的碎片；若因总RNA来源不同而存在组分差异的，可以适当增减酶用量及酶切时间，建议在30％范围内波动；
(3)酶切结束后可以于70℃加热15分钟将SLFN13-N失活，获得纯化的总mRNA。
步骤(1)中所述的样品为细胞样品或组织样品中的一种。
若步骤(1)中的样品特殊，总RNA可采用其它有效方法提取，尽量保证总RNA质量和完整性即可。
步骤(2)中所述的SLFN13为SLFN13全长或SLFN13的N端结构域中的一种。
所述SLFN13的N端结构域(统称为SLFN13-N)为人源SLFN13的氨基酸序列1-355(hSLFN13-N，Gene ID:146857)或大鼠SLFN13的氨基酸序列1-353(rSLFN13-N，Gene ID:303378)中的一种。
所述的人源SLFN13的Gene ID为146857，主要纯化使用其氨基酸序列的1-355。
所述的大鼠SLFN13的Gene ID为303378，主要纯化使用其氨基酸序列的1-353。
所述的SLFN13的N端结构域，可通过以下的表达纯化方法制备获得：
将SLFN13的N端结构域(人源SLFN13的氨基酸序列1-355，hSLFN13-N，Gene ID:146857和大鼠SLFN13的氨基酸序列1-353，rSLFN13-N，Gene ID:303378；统称为SLFN13-N)分别构建到pET28载体，测序验证正确后，将质粒转化入Rossetta(DE3)表达菌株；挑取单克隆入100ml添加卡那霉素和氨苄霉素双抗的LB培养基中预培养，12-16hr后将菌液按照1：100的比例转入5L添加双抗的TB培养基中在37℃扩大培养，待OD到0.4-0.6时，降温至17℃，加入80uM IPTG诱导表达SLFN13-N蛋白；低温诱导表达16-20h后，离心收集菌体并破碎释放蛋白；SLFN13-N的N末端含有一个6×His-tag可以用镍基质进行亲和纯化，最后用凝胶柱分子筛分选出均质单一的蛋白组分，浓缩至约2ug/ul(50uM)备用，冻存于-80℃保存。纯化结果见图1。
作为优选的实施方式，若对mRNA的纯度有更加严格要求，在第(3)步将酶失活之后，结合相应的small RNA纯化试剂盒将酶切后的tRNA和rRNA碎片去除，得到高纯度的总mRNA。
本发明制备获得的总mRNA可直接用于后续建库实验。
本发明相对于现有技术具有如下的优点及效果：
本发明打破传统的RNA纯化观念，引入特异性的RNA内切酶将需要去除的RNA分子从总RNA中酶切去除，简便高效。该专利的优点可概括为以下几个方面：
1)成本低，易获得。该发明纯化过程中不需借助太多额外的RNA纯化介质，比如特异性的RNA结合基质、特殊的RNA纯化缓冲液等。最关键的步骤是纯化得到有活性的内切酶，该酶可以通过普通的大肠杆菌菌株表达，经镍基质亲和层析结合分子筛分离便可获得纯度在90％以上的蛋白。培养3L可诱导表达的菌株，纯化后大概可获得50mg蛋白(约12mM)，而该酶的在体外的酶切效率很高，4pmol酶在室温酶切1ug RNA底物大概仅需要10-20分钟。因此，纯化一次酶的时间短、成本低但可多次使用。
2)操作简便。省去了RNA纯化基质的使用，略过了基质平衡，RNA特异结合及洗脱等过程。只需一步将合适的酶量加入RNA酶切体系即可，酶处理阶段只需静置或简单旋转，不需要额外的人为监测，后续的RNA碎片可略过纯化步骤，因为很小片段的RNA碎片对分子量较大的mRNA建库不会产生太多干扰。整个过程易掌握，不易引入错误，即使新手也可以快速熟练操作。
3)节约时间。因为操作步骤和实验流程简单，该方法总体耗时少，既可缩短实验周期又利于保障样品稳定。即使要去除酶切碎片，结合一个small RNA提取试剂盒也可以在半小时内完成。
4)纯化效果好，可以选择性的一步去除tRNA和rRNA,保证总mRNA的纯度和完整性。该发明主要依赖特异性的内切酶对要排除的RNA组分酶切去除，鉴于该内切酶的酶切选择性和特异性，可以一次性的把总RNA中含量最多的 tRNA和rRNA酶切去除，相较其它纯化方法更彻底。由于该酶对单链RNA无酶切活性，因此可以尽可能的保证mRNA的完整性。
5)利于保证mRNA的稳定性。RNA在空气中易被降解，该发明方法中，因省略了上样洗脱等过程，大大减少了在实验操作过程中引入的mRNA降解几率，利于保证纯化得到的总mRNA的质量。
附图说明
图1是hSLFN13-N和rSLFN13-N纯化后从单体蛋白的洗脱峰对应的收集管中分别制样进行SDS-PAGE分析的结果图。
图2是SLFN13-N对tRNA的选择性酶切后进行尿素胶电泳分析的结果。
图3是SLFN13-N的酶切活性位点确定及其对体内成熟tRNA的酶切活性检测结果，图a和图b分别为hSLFN13和rSLFN13及其相关突变对293T细胞中提取的small RNA的酶切效果。图c和图d分别为hSLFN13和rSLFN13及其相关突变对HeLa细胞中提取的small RNA的酶切效果。Small RNA中主要包含tRNA和5S和5.8S rRNA。
图4：Northern blot方法验证SLFN13-N对体内提取的总RNA中的tRNA和rRNA的酶切活性的检测结果。图a-c分别为用特异性针对tRNASer,tRNAGly和tRNALys的探针检测SLFN13对这三种成熟tRNA的酶切情况。图d是用5S rRNA的探针检测SLFN13对5S rRNA的酶切情况。
图5：SLFN13-N对细胞内提取的总RNA中rRNA的酶切结果。图a为SLFN13(hSLFN13和rSLFN13)及其相关突变对293T细胞内提取的总RNA的酶切情况。图b为SLFN13(hSLFN13和rSLFN13)及其相关突变对HeLa细胞内提取的总RNA的酶切情况。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例1 SLFN13的表达纯化
将SLFN13的N端结构域(人源SLFN13的氨基酸序列1-355，hSLFN13-N，Gene ID:146857和大鼠SLFN13的氨基酸序列1-353，rSLFN13-N，Gene ID:303378；统称为SLFN13-N)分别构建到pET28载体，测序验证正确后，将质粒转化入Rossetta(DE3)表达菌株；挑取单克隆入100ml添加卡那霉素和氨苄霉素双抗的LB培养基中预培养，12-16hr后将菌液按照1：100的比例转入5L添加双抗的TB培养基中在37℃扩大培养，待OD到0.4-0.6时，降温至17℃，加入80uM IPTG诱导表达SLFN13蛋白；低温诱导表达16-20h后，离心收集菌体并破碎释放蛋白；SLFN13-N的N末端含有一个6×His-tag可以用镍基质进行亲和纯化，最后用凝胶柱分子筛分选出均质单一的蛋白组分，浓缩至约2ng/ul(50uM)备用，冻存于-80℃保存。hSLFN13-N和rSLFN13-N的纯化结果见图1；由图可以看出，蛋白的纯度均在90％以上。
实施例2
本发明提供了一种用SLFN13从总RNA中纯化总mRNA的方法，具体步骤如下：
(1)总RNA提取：细胞或组织样品等可以利用传统的TRIzol-氯仿方法提取完整的总RNA(若样品特殊可以考虑其它适用方法，尽量保证总RNA质量和完整性即可)；
(2)tRNA与rRNA的酶切。根据从样品中提取的总RNA量及其tRNA、rRNA和mRNA的大概含量比计算需要加入的SLFN13内切酶的量和酶切时间；以总RNA中tRNA、rRNA和mRNA的含量分别为12％、83％和3％为例，若取 10ul浓度为1ug/ul的总RNA，加入1ul 50uM的SLFN13，加2ul 10×酶切缓冲液，加7ul ddH2O配成20ul的酶切体系(即每ug总RNA用5pmol SLFN13进行酶切，提供的酶浓度为50uM)；10×酶切缓冲液的成分为：400mM Tris-HCl(pH 8.0),200mM KCl,40mM MgCl2和20mM DTT；室温孵育酶切30min即可；图2-图5已经证明SLFN13-N可以特异性的酶切总RNA中的tRNA和rRNA，直至切为100nt以内的碎片为止。若因总RNA来源不同而存在组分差异的，可以适当增减酶用量，建议在30％范围内波动。
(3)酶切结束后可以于70℃加热15分钟将SLFN13-N失活；
(4)若对mRNA的纯度有更加严格要求的可以在第(3)步之后，结合相应的small RNA纯化试剂盒将酶切后的tRNA和rRNA碎片去除，得到高纯度的mRNA再进行建库步骤。
图2是SLFN13-N对tRNA的选择性酶切。如图所示将SLFN13-N与不同类型的核酸底物在体外孵育酶切30分钟后进行尿素胶电泳分析，结果显示SLFN13-N仅特异性的对tRNA进行酶切。
图3：SLFN13-N的酶切活性位点确定及其对体内成熟tRNA的酶切活性。为了更好的了解SLFN13-N的酶切性质，我们确定了其酶切活性位点并验证了相关蛋白对细胞内提取的成熟的tRNA的酶切活性。我们分别从HEK-293T细胞和HeLa中提取了tRNA含量在90％以上的small RNA作为底物进行酶切验证，结果表明SLFN13-N对细胞内提取的tRNA和体外转录的tRNA具有类似的酶切活性和酶切性质。
图4：Northern blot方法验证SLFN13-N对体内提取的总RNA中的tRNA和rRNA的酶切活性。分别设计了针对于tRNASer、tRNAGly、tRNALys和5S rRNA的探针并在5’端用P32进行标记，SLFN13-N与细胞内提取的总RNA孵 育反应后跑胶分离并转膜，分别用相应探针与酶切产物进行杂交。结果显示，SLFN13-N对细胞内提取的总RNA中的tRNA和5S rRNA具有明显的酶切活性，随时间和酶量增加，5S rRNA可被酶切为碎片。
图5：SLFN13-N对体内提取的总RNA中的rRNA的酶切活性。SLFN13-N及其相关酶活突变蛋白分别与从HEK-293T细胞和HeLa细胞中提取的总RNA孵育反应，随酶浓度升高酶切效果明显增强，随时间增加rRNA可被逐渐切为碎片。
该发明打破传统的RNA纯化观念，引入特异性的RNA内切酶将tRNA和rRNA分子从总RNA中酶切去除，简便高效。该专利的优点可概括为以下几个方面：
1)成本低，易获得。该发明纯化过程中不需借助太多额外的RNA纯化介质，比如特异性的RNA结合基质、特殊的RNA纯化缓冲液等。最关键的步骤是纯化得到有活性的内切酶，该酶可以通过普通的大肠杆菌菌株表达，经镍基质亲和层析结合分子筛分离便可获得纯度在90％以上的蛋白。培养3L可诱导表达的菌株，纯化后大概可获得50mg蛋白(约12mM)，而该酶的在体外的酶切效率很高，4pmol酶在室温酶切1ug RNA底物大概仅需要10-20分钟。因此，纯化一次酶的时间短、成本低但可以多次使用。
2)操作简便。省去了纯化基质的使用，略过了基质平衡，RNA特异结合及洗脱等过程。只需一步将合适的酶量加入RNA酶切体系即可，酶处理阶段只需静置或简单旋转，不需要额外的人为监测，后续的RNA碎片可略过纯化步骤，因为很小片段的RNA碎片对分子量较大的mRNA建库不会产生太多干扰。整个过程易掌握，不易引入错误，即使新手也可以快速熟练操作。
3)节约时间。因为操作步骤和实验流程简单，该方法总体耗时少，既可缩短实验周期又利于保障样品稳定。即使要去除酶切碎片，结合一个small RNA提取试剂盒也可以在半小时内完成。
4)纯化效果好，可以选择性的一步去除tRNA和rRNA,保证mRNA的纯度和完整性。该发明主要依赖特异性的内切酶对要排除的RNA组分酶切去除，鉴于该内切酶的酶切选择性和特异性，可以一次性的把总RNA中含量最多的tRNA和rRNA酶切去除，相较其它纯化方法更彻底。由于该酶对单链RNA无酶切活性，因此可以尽可能的保证mRNA的完整性。
5)利于保证mRNA的稳定性。RNA在空气中易被降解，该发明方法中，因省略了上样洗脱等过程，大大减少了在实验操作过程中引入的mRNA降解几率，利于保证纯化得到的总mRNA的质量。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (9)

  1. 一种用SLFN13从总RNA中纯化总mRNA的方法，其特征在于：具体步骤如下：
    (1)总RNA提取：利用传统的TRIzol-氯仿方法从样品中提取完整的总RNA；
    (2)tRNA与rRNA的酶切：以纯化10ug总RNA为例，取10ul浓度为1ug/ul的总RNA，加入1ul 50uM的SLFN13，加2ul 10×酶切缓冲液，加7ul ddH2O配成20ul的酶切体系；10×酶切缓冲液的成分为：400mM Tris-HCl(pH 8.0),200mM KCl,40mM MgCl2和20mM DTT；室温孵育酶切30min即可；
    (3)酶切结束后可以于70℃加热15分钟将SLFN13-N失活，获得纯化的总mRNA。
  2. 根据权利要求1所述的用SLFN13从总RNA中纯化总mRNA的方法，其特征在于：步骤(1)中所述的样品为细胞样品或组织样品中的一种。
  3. 根据权利要求1所述的用SLFN13从总RNA中纯化总mRNA的方法，其特征在于：若步骤(1)中的样品特殊，总RNA可采用其它有效方法提取。
  4. 根据权利要求1所述的用SLFN13从总RNA中纯化总mRNA的方法，其特征在于：步骤(2)中所述的SLFN13为SLFN13全长或SLFN13的N端结构域中的一种。
  5. 根据权利要求4所述的用SLFN13从总RNA中纯化总mRNA的方法，其特征在于：所述SLFN13的N端结构域为人源SLFN13的氨基酸序列1-355或大鼠SLFN13的氨基酸序列1-353中的一种。
  6. 根据权利要求5所述的用SLFN13从总RNA中纯化总mRNA的方法，其特征在于：所述的人源SLFN13的Gene ID为146857，主要纯化使用其氨基酸序列的1-355。
  7. 根据权利要求5所述的用SLFN13从总RNA中纯化总mRNA的方法，其特征在于：所述的大鼠SLFN13的Gene ID为303378，主要纯化使用其氨基酸序列的1-353。
  8. 根据权利要求4所述的用SLFN13从总RNA中纯化总mRNA的方法，其特征在于：所述的SLFN13的N端结构域，可通过以下的表达纯化方法制备获得：
    将SLFN13的N端结构域构建到pET28载体，测序验证正确后，将质粒转化入Rossetta(DE3)表达菌株；挑取单克隆入100ml添加卡那霉素和氨苄霉素双抗的LB培养基中预培养，12-16hr后将菌液按照1：100的比例转入5L添加双抗的TB培养基中在37℃扩大培养，待OD到0.4-0.6时，降温至17℃，加入80uM IPTG诱导表达SLFN13-N蛋白；低温诱导表达16-20h后，离心收集菌体并破碎释放蛋白；SLFN13-N的N末端含有一个6×His-tag可以用镍基质进行亲和纯化，最后用凝胶柱分子筛分选出均质单一的蛋白组分，浓缩至约2ug/ul备用，冻存于-80℃保存。
  9. 根据权利要求1所述的用SLFN13从总RNA中纯化总mRNA的方法，其特征在于：若对mRNA的纯度有更加严格要求，在第(3)步将酶失活之后，结合相应的small RNA纯化试剂盒将酶切后的tRNA和rRNA碎片去除，得到更高纯度的总mRNA。
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