4-Thiouridine
(Synonyms: 4-硫代尿苷) 目录号 : GC42474
4-Thiouridine 4-硫脲(4sU)是核糖核苷类似物，可用于RNA分析和(m)RNA标记。
Cas No.:13957-31-8
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
一、 4-thiouridine标记方案[1]：
4-thiouridine 溶液：用4-thiouridine粉末在ddH2O中配制成50mM的原液，过滤除菌。将溶液保存在- 20℃，避光，每份100μl。
1． 1.5 × 106 2fTGH细胞在4-thiouridine(10µM)下培养，裂解，提取总RNA，用于体内代谢标记。
2． 总RNA 50μg用biotin-HPDP (1mg/ml；2μl/μg RNA)在生物素化缓冲液 (100mM Tris, 10mM EDTA, pH 7.4, 1μl/μg RNA)中室温下处理1.5h。
3． 加入等量氯仿，混合，与生物素化RNA孵育3min。混合物在 pre-spun Phase Trap Gel Heavy Tubes中分离(5min, 16000rpm)。
4． 为了沉淀RNA和去除未掺入的biotin-HPDP，在水相中加入1/10体积的5M NaCl和等体积的异丙醇，离心(20min, 16000rpm)。
5．Pellet在等体积的75%乙醇中洗涤，离心(10min, 16000rpm)。
6． 将RNA重悬于100μl RNase-free 的水中。
7． 将未标记的RNA和4-thiouridine标记的RNA加热至65℃，10min，在冰上冷却5min。RNA与75μl链亲和素包被的磁珠旋转孵育15min。
8． 将反应体积施加于μMACs柱上，置于OctoMACS分离器磁架中，用900μl μMACs洗涤缓冲液(100mM Tris, 10mMEDTA, 1M NaCl, 0.1% Tween-20, pH 7.5)平衡。
9． 用μMACS洗涤缓冲液洗涤色谱柱。
10. 4-thiouridine-biotin-streptavidin标记的RNA用700μl 含有二硫赤藓糖醇(100mM)的RLT裂解缓冲液洗脱。
11. 用PeqGOLD总RNA试剂盒回收4-thiouridine标记RNA。
12. 4-thiouridine标记RNA用含溴化乙啶(37.5μg/100ml)的1.5%琼脂糖凝胶分离。
13. 用AIDA软件对紫外照射下RNA信号进行定量分析。
本程序仅提供指导，请根据您的具体需求进行修改。
References:
[1]. Burger K, Mühl B, et,al. 4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response. RNA Biol. 2013 Oct;10(10):1623-30. doi: 10.4161/rna.26214. Epub 2013 Sep 4. PMID: 24025460; PMCID: PMC3866244.
4-Thiouridine(4sU) is a ribonucleoside analogue that can be used for RNA analysis and (m)RNA labeling[1]. 4-Thiouridine is a sulfur-containing uridine derivative in which the oxygen atom at the 4-position of the uridine ring is replaced with a sulfur atom. This modification is naturally found in certain RNA molecules, particularly in bacterial transfer RNA (tRNA), where it plays a role in stabilizing RNA structure and enhancing its function[2-3].
In laboratory applications, 4-thiouridine serves as a labeling tool to study RNA synthesis, metabolism, and localization[4-5]. Upon UV irradiation, s4U-labeled RNA can form covalent cross-links with adjacent molecules, such as proteins, which facilitates the analysis of RNA interactions and dynamics within cells[6].
4-Thiouridine can interfere with nucleolar integrity and trigger a nucleolar stress response. 4-Thiouridine triggers translocation of NPM1, stabilization of p53, and inhibition of proliferation [7].
References:
[1]. Dölken L, Ruzsics Z, et,al. High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA. 2008 Sep;14(9):1959-72. doi: 10.1261/rna.1136108. Epub 2008 Jul 24. PMID: 18658122; PMCID: PMC2525961.
[2]. Hafner M, Landthaler M, et,al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell. 2010 Apr 2;141(1):129-41. doi: 10.1016/j.cell.2010.03.009. PMID: 20371350; PMCID: PMC2861495.
[3]. Windhager L, Bonfert T, et,al. Ultrashort and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution. Genome Res. 2012 Oct;22(10):2031-42. doi: 10.1101/gr.131847.111. Epub 2012 Apr 26. PMID: 22539649; PMCID: PMC3460197.
[4]. Rädle B, Rutkowski AJ, et,al. Metabolic labeling of newly transcribed RNA for high resolution gene expression profiling of RNA synthesis, processing and decay in cell culture. J Vis Exp. 2013 Aug 8;(78):50195. doi: 10.3791/50195. PMID: 23963265; PMCID: PMC3854562.
[5]. Schwanhäusser B, Busse D, et,al. Global quantification of mammalian gene expression control. Nature. 2011 May 19;473(7347):337-42. doi: 10.1038/nature10098. Erratum in: Nature. 2013 Mar 07;495(7439):126-7. doi: 10.1038/nature11848. PMID: 21593866.
[6]. Sun W, Chen W. Metabolic Labeling of Newly Synthesized RNA with 4sU to in Parallel Assess RNA Transcription and Decay. Methods Mol Biol. 2018;1720:25-34. doi: 10.1007/978-1-4939-7540-2_3. PMID: 29236249.
[7]. Burger K, Mühl B, et,al. 4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response. RNA Biol. 2013 Oct;10(10):1623-30. doi: 10.4161/rna.26214. Epub 2013 Sep 4. PMID: 24025460; PMCID: PMC3866244.
4-Thiouridine 4-硫脲(4sU)是核糖核苷类似物，可用于RNA分析和(m)RNA标记[1]。4-Thiouridine是一种含硫的尿嘧啶衍生物，其中尿嘧啶环上4位的氧原子被硫原子取代。这种修饰在某些RNA分子中自然存在，特别是在细菌转移RNA (tRNA)中，它在稳定RNA结构和增强其功能方面发挥作用[2-3]。
在实验室应用中，4-Thiouridine可作为研究RNA合成、代谢和定位的标记工具[4-5]。在紫外线照射下，4-Thiouridine标记的RNA可以与邻近的分子(如蛋白质)形成共价交联，这有助于分析RNA在细胞内的相互作用和动力学[6]。
4-Thiouridine可以干扰核仁完整性并引发核仁应激反应。4-Thiouridine触发NPM1易位，稳定p53，抑制增殖[7]。
| Cas No. | 13957-31-8 | SDF | |
| 别名 | 4-硫代尿苷 | ||
| Canonical SMILES | OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C=CC(NC2=O)=S)O1 | ||
| 分子式 | C9H12N2O5S | 分子量 | 260.3 |
| 溶解度 | DMF: 10 mg/ml,DMSO: 10 mg/ml,Ethanol: 2 mg/ml,PBS (pH 7.2): 5 mg/ml | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 3.8417 mL | 19.2086 mL | 38.4172 mL |
| 5 mM | 0.7683 mL | 3.8417 mL | 7.6834 mL |
| 10 mM | 0.3842 mL | 1.9209 mL | 3.8417 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
4-Thiouridine-Enhanced Peroxidase-Generated Biotinylation of RNA
Chembiochem 2021 Jan 5;22(1):212-216.PMID:32864814DOI:10.1002/cbic.202000567.
Peroxidase-generated proximity labeling is in widespread use to study subcellular proteomes and the protein interaction networks in living cells, but the development of subcellular RNA labeling is limited. APEX-seq has emerged as a new method to study subcellular RNA in living cells, but the labeling of RNA still has room to improve. In this work, we describe 4-Thiouridine (s4 U)-enhanced peroxidase-generated biotinylation of RNA with high efficiency. The incorporation of s4 U could introduce additional sites for RNA labeling, enhanced biotinylation was observed on monomer, model oligo RNA and total RNA. Through the s4 U metabolic approach, the in vivo RNA biotinylation efficiency by peroxidase catalysis was also dramatically increased, which will benefit RNA isolation and study for the spatial transcriptome.
Gaining insight into transcriptome-wide RNA population dynamics through the chemistry of 4-Thiouridine
Wiley Interdiscip Rev RNA 2019 Jan;10(1):e1513.PMID:30370679DOI:10.1002/wrna.1513.
Cellular RNA levels are the result of a juggling act between RNA transcription, processing, and degradation. By tuning one or more of these parameters, cells can rapidly alter the available pool of transcripts in response to stimuli. While RNA sequencing (RNA-seq) is a vital method to quantify RNA levels genome-wide, it is unable to capture the dynamics of different RNA populations at steady-state or distinguish between different mechanisms that induce changes to the steady-state (i.e., altered rate of transcription vs. degradation). The dynamics of different RNA populations can be studied by targeted incorporation of noncanonical nucleosides. 4-Thiouridine (s4 U) is a commonly used and versatile RNA metabolic label that allows the study of many properties of RNA metabolism from synthesis to degradation. Numerous experimental strategies have been developed that leverage the power of s4 U to label newly transcribed RNA in whole cells, followed by enrichment with activated disulfides or chemistry to induce C mutations at sites of s4 U during sequencing. This review presents existing methods to study RNA population dynamics genome-wide using s4 U metabolic labeling, as well as a discussion of considerations and challenges when designing s4 U metabolic labeling experiments. This article is categorized under: RNA Methods > RNA Analyses in Cells RNA Turnover and Surveillance > Regulation of RNA Stability.
The influence of 4-Thiouridine labeling on pre-mRNA splicing outcomes
PLoS One 2021 Dec 13;16(12):e0257503.PMID:34898625DOI:10.1371/journal.pone.0257503.
Metabolic labeling is a widely used tool to investigate different aspects of pre-mRNA splicing and RNA turnover. The labeling technology takes advantage of native cellular machineries where a nucleotide analog is readily taken up and incorporated into nascent RNA. One such analog is 4-Thiouridine (4sU). Previous studies demonstrated that the uptake of 4sU at elevated concentrations (>50μM) and extended exposure led to inhibition of rRNA synthesis and processing, presumably induced by changes in RNA secondary structure. Thus, it is possible that 4sU incorporation may also interfere with splicing efficiency. To test this hypothesis, we carried out splicing analyses of pre-mRNA substrates with varying levels of 4sU incorporation (0-100%). We demonstrate that increased incorporation of 4sU into pre-mRNAs decreased splicing efficiency. The overall impact of 4sU labeling on pre-mRNA splicing efficiency negatively correlates with the strength of splice site signals such as the 3' and the 5' splice sites. Introns with weaker splice sites are more affected by the presence of 4sU. We also show that transcription by T7 polymerase and pre-mRNA degradation kinetics were impacted at the highest levels of 4sU incorporation. Increased incorporation of 4sU caused elevated levels of abortive transcripts, and fully labeled pre-mRNA is more stable than its uridine-only counterpart. Cell culture experiments show that a small number of alternative splicing events were modestly, but statistically significantly influenced by metabolic labeling with 4sU at concentrations considered to be tolerable (40 μM). We conclude that at high 4sU incorporation rates small, but noticeable changes in pre-mRNA splicing can be detected when splice sites deviate from consensus. Given these potential 4sU artifacts, we suggest that appropriate controls for metabolic labeling experiments need to be included in future labeling experiments.
4-Thiouridine Labeling to Analyze mRNA Turnover in Schizosaccharomyces pombe
Cold Spring Harb Protoc 2017 May 1;2017(5).PMID:28461655DOI:10.1101/pdb.prot091645.
Traditionally, the half-lives of mRNAs were measured after inhibition of transcription to allow decay of the preexisting population. The protocol presented here is a more recently developed strategy in which mRNA turnover is analyzed by measuring the decline in levels of newly synthesized RNA labeled with 4-Thiouridine (4sU) during a brief pulse. After RNA extraction, the 4sU is biotinylated and the labeled species are purified using streptavidin beads. DNA microarrays can then be used to compare this population with total RNA, allowing half-lives to be calculated.
A bifunctional chemical signature enabling RNA 4-Thiouridine enrichment sequencing with single-base resolution
Chem Commun (Camb) 2022 Jan 27;58(9):1322-1325.PMID:34985087DOI:10.1039/d1cc06080e.
Both sequence enrichment and base resolution are essential for accurate sequencing analysis of low-abundance RNA. Yet they are hindered by the lack of molecular tools. Here we report a bifunctional chemical signature for RNA 4-Thiouridine (4sU) enrichment sequencing with single-base resolution. This chemical signature is designed for specific 4sU labeling with two functional parts. One part is a distal alkynyl group for the biotin-assisted pulldown enrichment of target molecules via click chemistry crosslinking. The other part is a -NH group proximal to the pyrimidine ring of 4sU. It allows 4sU-to-cytosine transition during the polymerase-catalyzed extension reaction based on altering hydrogen-bonding patterns. Ultimately, the 4sU-containing RNA molecules can be enriched and accurately analyzed by single-base resolution sequencing. The proposed method also holds great potential to investigate transcriptome dynamics integrated with high-throughput sequencing.