RNA G-quadruplexes (rG4) are non-canonical secondary structures formed by guanine-rich RNA sequences through Hoogsteen hydrogen bonding. These structures consist of stacked planar G-quartets stabilized by monovalent cations like K⁺ (Fig.1). Dynamic rG4 folding and unfolding regulate RNA transcription[1], chromatin modifier recruitment[2], pre-miRNA processing[3], mRNA translation[4, 5], and mRNA stability[6]. In addition, rG4s co-regulate gene expression with RNA modifications such as m7G[3], o8G[7], and m6A[8, 9]. Dysregulation of rG4 formation is involved in stress response[7], cancer gene expression regulation[10, 11], and diseases such as α-synuclein aggregation in Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy[12].

Figure 1. In G-rich RNA sequence, four guanines may be bound by Hoogsteen bonds to form a G-quartet. G-quartets can be stacked to form RNA G-quadruplex (RG4)[13].

Arraystar rG4 microarray profiling technology accurately quantifies rG4 structures in the transcriptome, which includes key steps of in vivo dimethyl sulfate (DMS) treatment, in vitro RNA refolding, and affinity capture using anti-G4 antibody. Next, the captured rG4 containing RNAs are treated with AlkB demethylation to remove byproducts generated from the DMS treatment and to eliminate m1A/m3C-induced bias. Finally, the rG4-RNAs are quantified using highly sensitive Arraystar rG4 microarray. With these techniques, not only are the rG4s in living cells effectively captured and detected, but also the biases caused by DMS-induced modifications are eliminated, which significantly improves the accuracy and reliability of rG4 quantitative profiling results.
 

• DMS in vivo treatment and in vitro refolding to restore the authentic native rG4 structure.

• Highly selective and efficient BG4 antibody to enrich rG4 containing RNA fragments.

• AlkB treatment to remove biases induced by m1A and m3C modifications.

• Arraystar microarray to sensitively detect, profile, and analyze rG4-RNAs even at low abundance.

Arraystar rG4 Array profiling workflow 

Figure 1. Arraystar rG4 Array profiling workflow. Cultured cells are treated with DMS to methylate A, C, and G but not G in rG4. The isolated RNA is fragmented, denatured and renatured in vitro in the presence of K to enable refolding of only the prior in vivo rG4 regions protected from the m7G modification. The RNAs containing rG4 structures are immunoprecipitated with anti-G quadruplex antibody (BG4). The enriched RNAs are demethylated with AlkB and reverse transcribed into double-stranded cDNA with added T7 promoter. Fluorescent antisense cRNA is transcribed in vitro by T7 polymerase from the t7 promoter using Cy3-CTP dye substrate. The cRNA is hybridized to Arraystar rG4 Array for profiling rG4 regions in the transcriptome.

Arraystar Human rG4s Microarray Specifications

References

[1] Kwok CK et al. rG4-seq reveals widespread formation of G-quadruplex structures in the human transcriptome. Nat. Methods 13, 841–844 (2016).
[2] Yang SY et al. Transcriptome-wide identification of transient RNA G-quadruplexes in human cells. Nat. Chem. Biol. 14, 180–183 (2018).
[3] Yeung PY et al. Systematic evaluation and optimization of the experimental steps in RNA G-quadruplex structure sequencing. Sci. Rep. 9, 8091 (2019).
[4] Weng X et al. Keth-seq for transcriptome-wide RNA structure mapping. Nat. Chem. Biol. 16, 489–492 (2020).
[5] Hansel-Hertsch R et al. G-quadruplex structures mark human regulatory chromatin. Nat. Genet. 48, 1267–1272 (2016).
[6] 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).
[7] 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).
[8] 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).
[9] Guo JU et al. RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria. Science. Sep 23;353(6306):aaf5371 (2016).
[10] von Hacht A et al. Identification and characterization of RNA guanine-quadruplex binding proteins. Nucleic Acids Res. Jun;42(10):6630-44 (2014).
[11] Haeusler et al. C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature 507, 195–200 (2014).
[12] McRae EKS et al. Human DDX21 binds and unwinds RNA guanine quadruplexes. Nucleic Acids Res. Jun 20;45(11):6656-6668 (2017).
[13] 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).
[14] 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).
[15] Yu H et al. G4Atlas: a comprehensive transcriptome-wide G-quadruplex database. Nucleic Acids Res. Jan 6;51(D1):D126-D134 (2023).
[16] Bourdon S et al. QUADRatlas: the RNA G-quadruplex and RG4-binding proteins database. Nucleic Acids Res. Jan 6;51(D1):D240-D247 (2023).
[17] 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).
[18] Zhong HS et al. G4Bank: A database of experimentally identified DNA G-quadruplex sequences. Interdiscip Sci. Sep;15(3):515-523 (2023).
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RNA G-quadruplex (rG4) databases

References

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