Cyclohexyl “base pairs” stabilize duplexes and intensify pyrene fluorescence by shielding it from natural base pairs

Abstract

In this study, we investigated the stability and structure of artificial base pairs that contain cyclohexyl rings. The introduction of a single pair of isopropylcyclohexanes into the middle of DNA slightly destabilized the duplex. Interestingly, as the number of the "base pairs" increased, the duplex was remarkably stabilized. A duplex with six base pairs was even more stable than one containing six A-T pairs. Thermodynamic analysis revealed that changes in entropy and not enthalpy contributed to duplex stability, demonstrating that hydrophobic interactions between isopropyl groups facilitated the base pairing, and thus stabilized the duplex. NOESY of a duplex containing an isopropylcyclohexane-methylcyclohexane pair unambiguously demonstrated its "pairing" in the duplex because distinct NOEs between the protons of cyclohexyl moieties and imino protons of both of the neighboring natural base pairs were observed. CD spectra of duplexes tethering cyclohexyl moieties also showed a positive-negative couplet that is characteristic of the B-form DNA duplex. Taken together, these results showed that cyclohexyl moieties formed base pairs in the DNA duplex without severely disturbing the helical structure of natural DNA. Next, we introduced cyclohexyl base pairs between pyrene and nucleobases as an "insulator" that suppresses electron transfer between them. We found a massive increase in the quantum yield of pyrene due to the efficient shielding of pyrene from nucleobases. The cyclohexyl base pairs reported here have the potential to prepare highly fluorescent labeling agents by multiplying fluorophores and insulators alternately into DNA duplexes.

Pseudo Base Pairs that Exhibit High Duplex Stability and Orthogonality through Covalent and Non-covalent Interactions

“…Surprisingly, the non─ planar pairs drastically stabilized the duplex even though they have no aromatic rings. 37 T m of a DNA duplex containing one H─ H pair (H1a/H1b) was 44.7 ℃, which was slightly lower than that of the native duplex (47.7 ℃). However, T m increased to 47.1 ℃ when two H─ H pairs were inserted (H2a/H2b).…”

“…For example, quantum yield of a pyrene derivative in the DNA duplex was below 0.01, and almost no emission was observed (Figure 8b). 37 In contrast, a strong peak of pyrene emission appeared when six H─ H pairs were inserted. As a result, the uorescence quantum yield of pyrene increased to 0.32, which was several hundred times higher than that without H─ H pairs.…”

“…We con rmed the structure of the DNA duplex containing an H─ K pair using NMR spectroscopy. 37 Results revealed that the cyclohexane derivatives were located inside the DNA duplex despite their non─ planar structures. Surprisingly, the non─ planar pairs drastically stabilized the duplex even though they have no aromatic rings.…”

Effect of Methyl Group on Acyclic Serinol Scaffold for Tethering Dyes on the DNA Duplex Stability

“…Thed estabilisation resultingf rom cis isomerisation in the cluster motif was smaller than for the other motifs, because this motif can accept even non-planar molecules such as cyclohexane derivatives. [19] Nevertheless, in the cluster motif il-threoninol showed the maximum DT m .A ll these resultsi ndicatet hat il-threoninol will afford efficient photo-regulation of duplex formation.…”

“…When three azobenzenes were present (N‐3X/C) or when the azobenzenes were inserted in the dimer motif (N‐1X/C‐1X), the differences among the scaffolds were greater (Figure C), with i l ‐threoninol resulting in maximal Δ T m values. The destabilisation resulting from cis isomerisation in the cluster motif was smaller than for the other motifs, because this motif can accept even non‐planar molecules such as cyclohexane derivatives . Nevertheless, in the cluster motif i l ‐threoninol showed the maximum Δ T m .…”