Recognition of double-stranded DNA using energetically activated duplexes with interstrand zippers of 1-, 2- or 4-pyrenyl-functionalized O2′-alkylated RNA monomers

Abstract

Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and - more recently - engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2′-alkylated uridine monomers X–Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe-target duplexes (ΔTm/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics.

Synthesis and characterization of oligodeoxyribonucleotides modified with 2′-thio-2′-deoxy-2′-S-(pyren-1-yl)methyluridine

“…Consistent with our previous observations with other Invader chemistries, 6,7,12,14,15 duplexes with +1 interstrand zippers of S monomers are less stable than probes with other zipper arrangements (compare T m 's for S2:S5 relative to other probe duplexes, Table 4). The energetically activated nature of S2:S5 was verified through analysis of thermodynamic parameters, which were obtained from denaturation curves.…”

“…However, incubation of DH1 with S2:S5 in a HEPES buffer for 12-16 hours at ambient temperatures did not result in formation of slower-migrating recognition complexes on non-denaturing PAGE gels even at 500-fold molar probe excess (Figure 4). This contrasts the observations with O2:O5 14 and N2L:N5 15 , which result in ~50% dsDNA recognition when used at ~20-fold molar excess, but is consistent with the comparatively low dsDNA-targeting potential of S2:S5 as judged by the $\Delta G_{rec}^{293}$ values. A similar outcome was obtained when DH1 was annealed in the presence of S2:S5 followed by room temperature incubation (Figure S5), indicating that the recognition complex is not stable at these experimental conditions.…”

“…30 Probe duplexes with other zipper arrangements do not display prominent emission at λ em ~490 nm (Figure 3). Based on our previously published molecular modeling structures of O2:O5 , 14 we speculate that the two pyrene moieties of S2:S5 co-stack inside the duplex core leading to excimer formation, while the excimer emission of S1:S4 is due to pyrene stacking in the major groove as suggested for other probes with +2 zipper arrangements of intercalator-modified nucleotides. 31 …”

“…As it is frequently observed with intercalator-modified ONs, 14,17,26 mismatched DNA targets are less efficiently discriminated than with unmodified reference strands. While S2 and O2 display similar binding fidelity, significantly better discrimination is observed with N2 .…”

“…Indeed, much lower $\Delta G_{rec}^{293}$ values are observed for S2:S5 than for other probe duplexes ($\Delta G_{rec}^{293}$ trend: S2:S5< < S2:S4≤S1:S2<S1:S5 , Table 4). However, contrary to our initial expectations, S2:S5 is less activated for dsDNA recognition than O2:O5 ${(\Delta G_{rec}^{293}=-29\mathrm{kJ}∕\mathrm{mol})}^{14}$ or N2:N5 ${(\Delta G_{rec}^{293}=-40\mathrm{kJ}∕\mathrm{mol})}^{15}$, in large part because the probe-target duplexes are significantly less stable (Δ G 293 for S2 :cDNA and S5 :cDNA = −10 kJ/mol, Table 4, compared to Δ G 293 for O2 :cDNA, O5 :cDNA, N2 :cDNA and N5 :cDNA = −14, −12, −20 and −19 kJ/mol, respectively 14,15 ).…”