一、簡(jiǎn)介

RNA G-四聯(lián)體 (RNA G-quadruplexes，rG4)是由富含鳥(niǎo)嘌呤 (G) 的RNA序列通過(guò)Hoogsteen氫鍵形成的一種非經(jīng)典二級(jí)結(jié)構(gòu)。該結(jié)構(gòu)由堆疊的G-四分體平面構(gòu)成，并由K⁺等單價(jià)陽(yáng)離子穩(wěn)定（圖1）。rG4的動(dòng)態(tài)結(jié)構(gòu)轉(zhuǎn)變可調(diào)控RNA轉(zhuǎn)錄[1]、染色質(zhì)修飾因子募集[2]、miRNA前體加工[3]、mRNA翻譯[4, 5]以及 mRNA 穩(wěn)定性[6]。此外，rG4 還能與m7G [3]、o8G[7]和m6A[8, 9]等 RNA 修飾共同調(diào)控基因表達(dá)。rG4形成發(fā)生失調(diào)，則會(huì)影響應(yīng)激反應(yīng) [7]、癌癥基因表達(dá)調(diào)控 [10, 11]，并與帕金森病、路易體癡呆及多系統(tǒng)萎縮中發(fā)生的α-突觸核蛋白聚集有關(guān)[12]。

圖1. 在富含鳥(niǎo)嘌呤的RNA序列中，四個(gè)鳥(niǎo)嘌呤通過(guò)Hoogsteen鍵結(jié)合形成G-四分體平面，G-四分體平面堆疊形成RNA G-四聯(lián)體（RG4）[13]。

Arraystar rG4芯片技術(shù)可精準(zhǔn)定量轉(zhuǎn)錄組中的rG4 結(jié)構(gòu)。其中的關(guān)鍵步驟包括：體內(nèi)硫酸二甲酯 (Dimethyl sulfate, DMS) 處理、體外RNA重折疊，以及使用抗G4抗體（BG4）進(jìn)行親和性捕獲。隨后，將捕獲到的含有 rG4結(jié)構(gòu)的 RNA進(jìn)行去甲基化處理，以去除 DMS 處理所產(chǎn)生的副產(chǎn)物m1A/m3C，消除其造成的檢測(cè)干擾。最后，利用高靈敏度的Arraystar rG4 芯片探針對(duì)rG4-RNA進(jìn)行定量分析。Arraystar rG4芯片技術(shù)不僅能夠有效捕獲和檢測(cè)活細(xì)胞中的rG4，同時(shí)也消除了DMS誘導(dǎo)的修飾所產(chǎn)生的偏差，從而顯著提高了rG4 定量圖譜分析結(jié)果的準(zhǔn)確性和可靠性。

二、技術(shù)優(yōu)勢(shì)

• 體內(nèi)DMS處理與體外重折疊復(fù)現(xiàn)了真實(shí)的rG4結(jié)構(gòu)

• 使用高親和性的BG4抗體特異性富集含有rG4結(jié)構(gòu)的RNA

• Demthylation處理去除了DMS 處理的副產(chǎn)物m1A/m3C所造成的檢測(cè)干擾

• Arraystar 芯片可以靈敏的檢測(cè)rG4 RNA, 包括RNA測(cè)序無(wú)法準(zhǔn)確檢測(cè)的低豐度RNA  

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三、實(shí)驗(yàn)流程

圖2. rG4芯片實(shí)驗(yàn)流程圖。

1. 體內(nèi) DMS 處理

培養(yǎng)細(xì)胞經(jīng)硫酸二甲酯（DMS）處理，對(duì) RNA 中的腺嘌呤（A）、胞嘧啶（C）及非 rG4折疊區(qū)域的鳥(niǎo)嘌呤（G）進(jìn)行甲基化修飾。rG4 結(jié)構(gòu)內(nèi)的 G 堿基因空間位阻不受影響，從而保留其未甲基化狀態(tài)。

2. RNA 抽提與體外重折疊

分離總 RNA 并進(jìn)行片段化處理，隨后在含K⁺的緩沖體系中經(jīng)歷變性-復(fù)性過(guò)程。此步驟僅允許細(xì)胞內(nèi)原有的 rG4 區(qū)域特異性重折疊，而其他區(qū)域由于攜帶m7G修飾，無(wú)法形成穩(wěn)定結(jié)構(gòu)。

3. rG4 免疫沉淀

利用抗 G-四聯(lián)體抗體（BG4）對(duì)含 rG4 結(jié)構(gòu)的 RNA 進(jìn)行免疫沉淀（IP），實(shí)現(xiàn)目標(biāo)分子的特異性富集。

4. 去甲基化與反轉(zhuǎn)錄

富集的 RNA 經(jīng)去甲基化酶處理，清除 DMS 誘導(dǎo)的副產(chǎn)物（如 m¹A/m³C），消除檢測(cè)偏差。隨后通過(guò)反轉(zhuǎn)錄合成雙鏈 cDNA，并引入T7 啟動(dòng)子序列。

5. 熒光標(biāo)記 cRNA 合成

以 cDNA 為模板，T7 RNA 聚合酶催化體外轉(zhuǎn)錄反應(yīng)，摻入Cy3-CTP 熒光染料，生成帶Cy3 標(biāo)記的反義 cRNA。

6. 芯片雜交與數(shù)據(jù)分析

標(biāo)記的 cRNA 與Arraystar rG4 芯片雜交，通過(guò)熒光信號(hào)定量分析轉(zhuǎn)錄組中rG4 結(jié)構(gòu)的分布與豐度。

四、芯片參數(shù)

Arraystar 人類 rG4 芯片參數(shù)

RNA G-quadruplex (rG4) 數(shù)據(jù)庫(kù)

參考文獻(xiàn)

1. Yari H et al: LncRNA REG1CP promotes tumorigenesis through an enhancer complex to recruit FANCJ helicase for REG3A transcription. Nat Commun 2019, 10(1):5334.[PMID: 31767869]

2. Lee YW, Weissbein U, Blum R, Lee JT: G-quadruplex folding in Xist RNA antagonizes PRC2 activity for stepwise regulation of X chromosome inactivation. Mol Cell 2024, 84(10):1870-1885 e1879.[PMID: 38759625]

3. Pandolfini L et al: METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation. Mol Cell 2019, 74(6):1278-1290 e1279.[PMID: 31031083]

4. Arora A, Suess B: An RNA G-quadruplex in the 3' UTR of the proto-oncogene PIM1 represses translation. RNA Biol 2011, 8(5):802-805.[PMID: 21734463]

5. Song J, Perreault JP, Topisirovic I, Richard S: RNA G-quadruplexes and their potential regulatory roles in translation. Translation (Austin) 2016, 4(2):e1244031.[PMID: 28090421]

6. Rouleau S et al: 3' UTR G-quadruplexes regulate miRNA binding. RNA 2017, 23(8):1172-1179.[PMID: 28473452]

7. Ma Y et al: RNA G-Quadruplex within the 5'-UTR of FEN1 Regulates mRNA Stability under Oxidative Stress. Antioxidants (Basel) 2023, 12(2).[PMID: 36829835]

8. Yoshida A et al: Recognition of G-quadruplex RNA by a crucial RNA methyltransferase component, METTL14. Nucleic Acids Res 2022, 50(1):449-457.[PMID: 34908152]

9. Jara-Espejo M, Fleming AM, Burrows CJ: Potential G-Quadruplex Forming Sequences and N(6)-Methyladenosine Colocalize at Human Pre-mRNA Intron Splice Sites. ACS Chem Biol 2020, 15(6):1292-1300.[PMID: 32396327]

10. Anastasakis DG et al: Nuclear PKM2 binds pre-mRNA at folded G-quadruplexes and reveals their gene regulatory role. Mol Cell 2024, 84(19):3775-3789 e3776.[PMID: 39153475]

11. Kharel P, Ivanov P: PKM2-G-quadruplex interactions conspire to regulate the cancer transcriptome. Mol Cell 2024, 84(19):3574-3575.[PMID: 39366344]

12. Matsuo K et al: RNA G-quadruplexes form scaffolds that promote neuropathological alpha-synuclein aggregation. Cell 2024, 187(24):6835-6848 e6820.[PMID: 39426376]

13. Dumas L et al: G-Quadruplexes in RNA Biology: Recent Advances and Future Directions. Trends Biochem Sci 2021, 46(4):270-283.[PMID: 33303320]

14. Kwok CK et al. rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome. Nat. Methods 13, 841–844 (2016).

15. Yang SY et al. Transcriptome-wide identification of transient RNA G-quadruplexes in human cells. Nat. Chem. Biol. 14, 180–183 (2018).

16. Yeung PY et al. Systematic evaluation and optimization of the experimental steps in RNA G-quadruplex structure sequencing. Sci. Rep. 9, 8091 (2019).

17. Weng X et al. Keth-seq for transcriptome-wide RNA structure mapping. Nat. Chem. Biol. 16, 489–492 (2020).

18. Hansel-Hertsch R et al. G-quadruplex structures mark human regulatory chromatin. Nat. Genet. 48, 1267–1272 (2016).

19. Herviou P et al. hnRNP H/F drive RNA G-quadruplex-mediated translation linked to genomic instability and therapy resistance in glioblastoma. Nat. Commun. 11, 2661 (2020).

20. Simko EAJ et al. G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA. Nucleic Acids Res. 48, 7421–7438 (2020).

21. Bolduc F et al. The small nuclear ribonucleoprotein polypeptide A (SNRPA) binds to the G-quadruplex of the BAG-1 5'UTR. Biochimie 176, 122–127 (2020).

22. Guo JU et al. RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria. Science. Sep 23;353(6306):aaf5371 (2016).

23. von Hacht A et al. Identification and characterization of RNA guanine-quadruplex binding proteins. Nucleic Acids Res. Jun;42(10):6630-44 (2014).

24. Haeusler et al. C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507, 195–200 (2014).

25. McRae EKS et al. Human DDX21 binds and unwinds RNA guanine quadruplexes. Nucleic Acids Res. Jun 20;45(11):6656-6668 (2017).

26. Serikawa T et al. Comprehensive identification of proteins binding to RNA G-quadruplex motifs in the 5' UTR of tumor-associated mRNAs. Biochimie. Jan;144:169-184 (2018).

27. Herdy B et al. Analysis of NRAS RNA G-quadruplex binding proteins reveals DDX3X as a novel interactor of cellular G-quadruplex containing transcripts. Nucleic Acids Res. Nov 30;46(21):11592-11604 (2018).

28. Yu H et al. G4Atlas: a comprehensive transcriptome-wide G-quadruplex database. Nucleic Acids Res. Jan 6;51(D1):D126-D134 (2023).

29. Bourdon S et al. QUADRatlas: the RNA G-quadruplex and RG4-binding proteins database. Nucleic Acids Res. Jan 6;51(D1):D240-D247 (2023).

30. Qian SH et al. EndoQuad: a comprehensive genome-wide experimentally validated endogenous G-quadruplex database. Nucleic Acids Res. Jan 5;52(D1):D72-D80 (2024).

31. Zhong HS et al. G4Bank: A database of experimentally identified DNA G-quadruplex sequences. Interdiscip Sci. Sep;15(3):515-523 (2023).

32. Birney E et al: An overview of Ensembl. Genome Res. May;14(5):925-8 (2004).

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