Cytosine-rich oligonucleotides incorporating a non-nucleotide loop: A further step towards the obtainment of physiologically stable i-motif DNA
Graphical abstract
Introduction
i-Motifs, also known as i-tetraplexes, are unusual secondary structures of nucleic acids formed by four C-rich oligonucleotide tracts (CROs) thanks to the formation of N3 hemiprotonated cytosines base pairs (C-C+, A, Fig. 1) [1], [2], [3]. In an i-motif, a parallel duplex formed by C-C+ base pairs (B, Fig. 1) is combined in an antiparallel manner with a second similar duplex by intercalating cytosine base pairs, thus forming a tetrastranded structure (C, Fig. 1). Tetramolecular, bimolecular, and monomolecular i-motif complexes have been reported in the literature (C-F, Fig. 1) [4], [5], [6]. Since their formation requires the hemiprotonation of cytosines, most i-motif structures are stable only at pH values close to the pKa of cytosine's N3 (pKa ≈ 4.8 in water). However, studies have demonstrated that i-motifs can be obtained at neutral or nearly neutral pH [7], [8], [9]. i-Motif stability can depend on several factors. Apart from the pH, the non-cytosine nucleotides flanking the poly-C strands play an essential role in i-motif stabilization, especially for tetramolecular complexes. Also, the stability of bimolecular or unimolecular i-motif complexes strictly depends on the length and base composition of the loops [9], [10], [11], [12]. Chemical modifications of CROs and the crowding effect aimed to stabilize and enlarge the structural variety of the i-motif DNA have also been investigated [13], [14], [15], [16], [17], [18].
i-Motif structures, discovered for the first time in 1963 [19], have attracted considerable interest because of their possible implication in biological processes, such as the telomers and centromeres formation, and as transcriptional regulators [20], [21], [22]. It was reported that C-rich DNAs are bound by proteins, such as the poly-C-binding proteins (PCBP), and, interestingly, when structured in i-motif, they can bind proteins belonging to the heterogeneous nuclear ribonucleoprotein (hnRNP) family [23]. i-Motif-containing aptamers have been used to specifically bind cells by exploiting the peculiarities of cellular pHs [24], [25], [26], [27]. Furthermore, ultra-pH-responsive i-motif aptamers were employed in sensing technologies developed to specifically achieve imaging of different cancer types in vitro and in vivo by exploiting the slight differences in extracellular pH values of different cancers [28]. i-Motifs have been considered potential building blocks for constructing supramolecular assemblies in DNA nanotechnology [29], [30], [31], [32], [33]. Furthermore, based on i-motif structures, pH-responsive DNA nanomachines [34], hydrogels [35], [36], pH-stimulated drug-releasing carriers [37], and controlled nano-gold assemblies have been reported [38], [39].
Our group has been studying guanine-rich oligonucleotides (GROs) containing non-nucleotide linkers acting as long non-nucleotide loops for several years. Double-Ended Linkers (DELs) and Tetra-Ended Linkers (TELs) bearing at their ends parallel or antiparallel GROs were explored in the field of the G-quadruplex structures [40], [41], [42], [43], [44], [45]. We observed that DEL and TEL linkers greatly influence the G-quadruplex properties. We found a more rapid formation and increased thermal stability of DEL- and TEL-G-quadruplexes than the corresponding tetramolecular counterparts. Furthermore, DEL- and TEL-GROs showed attitudes towards forming G-quadruplex multimers having higher molecular weight [40], [46]. In the present study, we explore the effect of the insertion of the DEL non-nucleotide loop connecting two CROs on the formation of i-motif structures. The DEL insertion can be achieved by retaining the CRO orientation, thus producing 5′-CRO-3′-DEL-5′-CRO-3′, or introducing an inversion of strands polarity to obtain 5′-CRO-3′-DEL-3′-CRO-5′ or 3′-CRO-5′-DEL-5′-CRO-3′ (these latter indicated as DEL-3′-CRO2 or DEL-5′-CRO2, respectively). The inversion of polarity in DEL-CROs enlarges the number of possible i-motif topologies. However, only a few examples of this modification have been reported in the literature [47], [48], [49]. Without an inversion of polarity, only two bimolecular i-motif structures are allowed (D and E in Fig. 1). In structure D, the two lateral loops are on the same side of the i-motif scaffold, whereas in E the loops are on the opposite sides. DEL-CROs containing an inversion of polarity can lead to additional i-motif topologies in which the DEL loops (in blue) are located diagonally on the opposite ends of the i-motif (G and H, Fig. 1). Furthermore, if the DEL-linker has a suitable length, the structures I and L (Fig. 1), characterized by two DEL propeller loops, could also be obtainable.
Herein, we report a strategy in which the DEL (9, Scheme 1) acts as a non-nucleotide loop and contextually introduces an inversion of polarity in DEL-CROs 3–6 (Scheme 1). In DEL-d(AC4A)2 (3 and 4), two parallel oligodeoxynucleotides (ODNs) are connected to the two linker's arms by their 5′ (in 3) or 3′ ends (in 4). Analogously, in DEL-d(C6)2 (5 and 6), two parallel ODNs are connected to the linker by 5′ (in 5) or 3′ ends (in 6). The structural features of i-motif structures formed by 3–6 were determined in comparison with those of the corresponding structures formed by d(AC4A) and d(C6) (7 and 8 in Scheme 1, respectively). The obtainment, thermal stability, and molecularity of all i-motif species were assessed by CD, CD melting, PAGE, HPLC size exclusion chromatography, and 1H NMR experiments.
Section snippets
Materials
All reagents and solvents were obtained from commercial sources and used without further purification. Reagents and phosphoramidites for DNA syntheses were purchased from Glen Research (Sterling, VA, USA). The DEL-linker 9 and the (CPG)-sulphone-resin 1 were purchased from Glen Research (Sterling, VA, USA). The ODNs were assembled over a PerSeptive Biosystems (Framingham, MA, USA) Expedite DNA synthesizer, using the phosphoramidite chemistry. The HPLC analyses and purifications were carried out
Results and discussion
DEL-CROs 3–6 were synthesized through a solid-phase automated DNA synthesizer, using the commercially available controlled pore glass (CPG)-sulphone-resin 1 (Scheme 1) and the phosphoramidite linker 9 to obtain the support 2. On support 2, the CRO syntheses were performed using 3′ or 5′ phosphoramidite building blocks to obtain the DEL-CROs 4 and 6, and 3 and 5, respectively [46]. The natural d(AC4A) (7) and d(C6) (8) were synthesized by standard phosphoramidite protocols. After their
Conclusion
DNA oligonucleotides represent an attractive building material to be used in supramolecular assembly and nanotechnology. DNA duplex, triplex, and G-quadruplex secondary structures have been extensively studied to construct desirable molecular or supramolecular scaffolds. DNA i-motif secondary structures enlarge the capability of the DNA toolbox to build new molecular architectures. It is noteworthy that i-motifs are pH-sensitive and can induce conformational changes as the pH varies. Some of
CRediT authorship contribution statement
Francesca Greco: Conducting the experiment; Investigation; Data curation
Maria Marzano: Assisting the experiment; Investigation
Andrea P. Falanga: Assisting the experiment; Editing
Monica Terracciano: Data curation; Funding acquisition
Gennaro Piccialli: Funding acquisition; Writing original draft
Giovanni N. Roviello: Editing; Data curation
Stefano D’Errico: Data curation
Nicola Borbone: Supervision; Funding acquisition; Data curation; Writing final draft
Giorgia Oliviero: Supervision; Funding
Declaration of competing interest
The authors declare that they have no conflict of interests.
Acknowledgments
This work was supported by the institutional financial support from Ministero dell'Università e della Ricerca (MUR) to ISBE Italy within the Italian Roadmap for ESFRI Research Infrastructures, and by the Department of Pharmacy - University of Naples Federico II grant “Sostegno allo Sviluppo della Ricerca Dipartimentale”. Dr. M. Terracciano acknowledges financial support from MUR's grant PON-AIM RTDA_L1 (AIM 1873131-2). The authors are grateful to Dr. C. Cassiano of the “Laboratorio di Analisi
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2023, International Journal of Biological Macromolecules