Hsa‐miR‐1587 G‐quadruplex formation and dimerization induced by NH4+, molecular crowding environment and jatrorrhizine derivatives
Graphical abstract
Introduction
G-quadruplexes widely exist in genome and transcriptome, such as telomeres, oncogenic promoters, immunoglobulin switches, mutational hot spot sequences, introns and the untranslated regions of mRNA [1], [2], [3]. With the increasing evidence of the existence and regulatory role of G-quadruplexes in biological processes [4], G-quadruplex structures have been considered as promising therapeutic targets and different kinds of G-quadruplex ligands have been developed [5], [6], [7], [8].
DNA G-quadruplexes are highly polymorphic regarding to the relative strand orientations (parallel and antiparallel), glycosidic conformations of guanine bases (anti and syn), groove widths (wide, medium and narrow) and intervening loops (double-chain-reversal, diagonal and lateral loops) [9], [10]. Based on these conformational features, a discrete DNA G-quadruplex structure generally has three kinds of typical folding topologies: parallel, hybrid and anti-parallel. Irregular folding topologies are also reported, such as snapback configurations [11], [12], [13], bulges in interrupted G-column [14], [15] and V-shaped loops connecting two or three G-quartets [16], [17], [18]. Guanine-rich sequences with four, two or one G-tracts could form intramolecular, bimolecular or tetramolecular G-quadruplexes. Interestingly, sequences with four G-tracts capable of forming intramolecular G-quadruplexes are also reported to form bimolecular G-quadruplexes, such as the bimolecular c-kit2 [19] and N-myc [20] G-quadruplexes with six stacking G-quartets formed in K+ solution. Moreover, DNA G-quadruplexes can also form high-order structures, interlocked G-quadruplexes [21], [22], [23], [24] and end-to-end stacking G-quadruplexes [25], [26], [27], [28], [29]. Several factors including flanking sequences, the type and concentration of cations and molecular crowding environment have been reported to influence the stacking of DNA G-quadruplexes [25], [26], [27], [28], [29], [30]. Besides, a perylene derivative, PIPER, has been reported to accelerate the association of bimolecular and tetramolecular G-quadruplexes [31].
In contrast to the polymorphic DNA G-quadruplex, RNA G-quadruplex is normally monomorphic. The 2′ hydroxyl groups in RNA limited the glycosidic conformations of guanine bases to adopt anti conformation, so that RNA G-quadruplex can only exist in parallel conformation with parallel strand orientations and double-chain reversal loops [32]. Moreover, reports about high-order RNA G-quadruplexes are relatively fewer [33], [34], [35], [36], [37], [38] and factors that influence high-order RNA G-quadruplex formation are still under to be investigated.
Electrospray ionization mass spectrometry (ESI-MS) has been widely used as a robust and convenient technique in the determination of molecularity of biological macromolecules in solution [39], [40]. Furthermore, several academic groups have shown that the high-order nucleic acids and their non-covalent complexes could be preserved and detected in mass spectrometer. Both intramolecular and intermolecular G-quadruplexes could preserve their structures and stably exist in ESI-MS conditions due to the formation of eight hydrogen bonds in G-quartets and electrostatic interactions between central cations and G-quartets [41], [42]. In this study, ESI-MS, tandem MS and high resolution MS were utilized to study the formation and properties of a RNA G-quadruplex formed by miR-1587, r(5′-UUGGGCUGGGCUGGGUUGGG-3′), a guanine-rich human mature microRNA [43]. Utilizing these methods, herein we further demonstrated that the miR‐1587 G‐quadruplex formed dimeric G-quadruplex in high concentration of NH4+ or molecular crowding environment with high thermal stability. Specifically, we discovered that two jatrorrhizine derivatives could also induce the formation of the dimeric G-quadruplex. Our study demonstrated the formation of a human mature microRNA G-quadruplex and investigated several factors that induced the miR‐1587 G‐quadruplex dimerization, enhancing our understanding of dimeric RNA G-quadruplex formation.
Section snippets
Materials
The oligonucleotide of miR-1587, r(5′-UUGGGCUGGGCUGGGUUGGG-3′), was purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). The sample was dissolved in ultrapure water (18.3 MΩ cm) to prepare stock solutions of 100 μM.
ESI mass spectrometry
The ESI-MS experiments were performed on a Finnigan LCQ Deca XP Plus ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). The instrument was used in the negative ion mode, with the capillary voltage set to 3.0 kV, the sheath gas flow rate set to 25 arb, and the
The formation of miR-1587 monomeric G-quadruplex
We performed ESI-MS and CD experiments to investigate the formation and property of miR-1587 G-quadruplex. It is known that ammonium ions can induce and stabilize the formation of G-quadruplex, and n adducted ammonium ions of the peaks in ESI-MS spectra indicated the formation of n+1G-quartets in the G-quadruplex [44], [45]. As shown in Fig. 1a, the miR-1587 sequence (abbreviated to S) displayed multiple peaks with eight to thirteen negative charges ([S]8- to [S]13-) in the absence of NH4OAc,
Conclusions
In this study, we demonstrated the formation and property of the miR‐1587 G‐quadruplex. Importantly, high concentration of NH4+, molecular crowding environments and two jatrorrhizine derivatives were discovered to be capable of inducing the formation of dimeric miR‐1587 G‐quadruplex. The dimeric G-quadruplex was formed through 3′-to-3′ stacking of two monomeric miR‐1587 G‐quadruplex subunits with one ammonium ion located between the interfaces, which contributed to the high thermal stability of
Acknowledgment
This work was supported by the National Natural Science Foundation of China (21572016, 21372021).
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