Cytosine-rich oligonucleotides incorporating a non-nucleotide loop: A further step towards the obtainment of physiologically stable i-motif DNA

Highlights

• New i-motif DNA structures incorporating a non-nucleotide loop (DEL linker) are presented.

• The DEL linker allowed to obtain very stable i-motif structures at pH 4.5.

• One of the synthesized DEL-i-motif structures proved stable even at pH 7.0.

• DEL-i-motif structures can form higher-order aggregates at pH 4.5.

Abstract

i-Motifs, also known as i-tetraplexes, are secondary structures of DNA occurring in cytosine-rich oligonucleotides (CROs) that recall increasing interest in the scientific community for their relevance in various biological processes and DNA nanotechnology. This study reports the design of new structurally modified CROs, named Double-Ended-Linker-CROs (DEL-CROs), capable of forming stable i-motif structures. Here, two C-rich strands having sequences d(AC₄A) and d(C₆) have been attached, in a parallel fashion, to the two linker's edges by their 3′ or 5′ ends. The resulting DEL-CROs have been investigated for their capability to form i-motif structures by circular dichroism, poly-acrylamide gel electrophoresis, HPLC–size-exclusion chromatography, and NMR studies. This investigation established that DEL-CROs could form more stable i-motif structures than the corresponding unmodified CROs. In particular, the i-motif formed by DEL-5′-d(C₆)₂ resulted stable enough to be detected even at near physiological conditions (37 °C, pH 7.0). The results open the way to developing pH-switchable nanocarriers and aptamers based on suitably functionalized DEL-CROs.

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 pK_a of cytosine's N3 (pK_a ≈ 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.