New G-Quadruplex-Forming Oligodeoxynucleotides Incorporating a Bifunctional Double-Ended Linker (DEL): Effects of DEL Size and ODNs Orientation on the Topology, Stability, and Molecularity of DEL-G-Quadruplexes

Abstract

G-quadruplexes (G4s) are unusual secondary structures of DNA occurring in guanosine-rich oligodeoxynucleotide (ODN) strands that are extensively studied for their relevance to the biological processes in which they are involved. In this study, we report the synthesis of a new kind of G4-forming molecule named double-ended-linker ODN (DEL-ODN), in which two TG₄T strands are attached to the two ends of symmetric, non-nucleotide linkers. Four DEL-ODNs differing for the incorporation of either a short or long linker and the directionality of the TG₄T strands were synthesized, and their ability to form G4 structures and/or multimeric species was investigated by PAGE, HPLC⁻size-exclusion chromatography (HPLC⁻SEC), circular dichroism (CD), and NMR studies in comparison with the previously reported monomeric tetra-ended-linker (TEL) analogues and with the corresponding tetramolecular species (TG₄T)₄. The structural characterization of DEL-ODNs confirmed the formation of stable, bimolecular DEL-G4s for all DEL-ODNs, as well as of additional DEL-G4 multimers with higher molecular weights, thus suggesting a way towards the obtainment of thermally stable DNA nanostructures based on reticulated DEL-G4s.

Keywords: CD; DEL-ODNs; G-quadruplexes; NMR; TEL-ODNs; double-ended linkers; size-exclusion chromatography; supramolecular G-quadruplexes.

Scheme 1

General synthetic route for compounds D1L,S, D2L,S and TEL1L,S, TEL2L,S. (i) Coupling with the phosphoramidite linker 4 or 5; (ii) two sequential couplings with the phosphoramidite linker 4 or 5; (iii) solid-phase DNA synthesis by phosphoramidite chemistry; iv) annealing procedure in Na⁺- or K⁺-containing buffer.

Figure 1

Circular dichroism spectra recorded at 5 °C of D1L,S and D2L,S annealed in 100 mM K⁺ buffer.

Figure 2

PAGE analyses of complexes formed by DEL-(TG₄T)₂ ODNs (D1L,S and D2L,S) in comparison with the (TG₄T)₄ G4 (A) and with the complexes formed by TEL-(TG₄T)₄ ODNs (TEL1L,S and TEL2L,S) (B). All samples were annealed in 100 mM K⁺ buffer.

Figure 3

HPLC–SEC chromatograms of the complexes formed by DEL-(TG₄T)₂ (D1L,S, D2L,S) and TEL-(TG₄T)₄ (TEL1L,S and TEL2L,S) in comparison with the (TG₄T)₄ G4 (Q₁) and with the Q_n G-wires.

Figure 4

Schematic representation of the parallel G4s obtainable from the self-assembly of DEL-ODNs. The first three complexes, having the least MW, are shown on the left. The hypothetical topology of G4 supramolecular nanostructures based on reticulated parallel DEL-G4s is shown on the right.

Figure 5

Downfield region of the water-suppressed NMR spectra of D1L,S and D2L,S annealed in 100 mM K⁺ buffer and recorded at 25, 45 and 85 °C.

Figure 6

HPLC–SEC chromatograms of: (A) complexes formed by D2L; (B) reinjection of the higher MW species formed by D2L collected as shown in panel A; (C) reinjection of the purified DEL-Q₁ complex formed by D2L collected as shown in panel A.

Figure 7

CD spectra of DEL-Q₁ and higher MW species obtained by HPLC–SEC fractionation of D2L (100 mM K⁺-containing buffer, 5 °C).