pH-Dependent mismatch discrimination of oligonucleotide duplexes containing 2′-deoxytubercidin and 2- or 7-substituted derivatives: protonated base pairs formed between 7-deazapurines and cytosine

Abstract

Oligonucleotides incorporating 2′-deoxytubercidin (1a), its 2-amino derivative 2a and related 2-, or 7-substituted analogs (1d, 2b–d, 3 and 4) are synthesized. For this purpose, a series of novel phosphoramidites are prepared and employed in solid-phase synthesis. Hybridization experiments performed with 12mer duplexes indicate that 7-halogenated nucleosides enhance the duplex stability both in antiparallel and parallel DNA, whereas 2-fluorinated 7-deaza-2′-deoxyadenosine residues destabilize the duplex structure. The 7-deazaadenine nucleosides 1a, 1d and their 2-amino derivatives 2a–d form stable base pairs with dT but also with dC and dG. The mispairing with dC is pH-dependent. Ambiguous base pairing is observed at pH 7 or under acid conditions, whereas base discrimination occurs in alkaline medium (pH 8.0). This results from protonated base pairs formed between 1a or 2a and dC under neutral or acid condition, which are destroyed in alkaline medium. It is underlined by the increased basicity of the pyrrolo[2,3-d]pyrimidine nucleosides over that of the parent purine compounds (pKa values: 1a = 5.30; 2a = 5.71; dA = 3.50).

Fluorine Substituted Adenosines As Probes of Nucleobase Protonation in Functional RNAs

“…This finding is consistent with the observation that the normal triphosphate of 2F-A can be readily incorporated by in vitro transcription, and that RNA duplexes containing this modification are only modestly destabilized42. A NAIM approach is particularly attractive for 2F-A since fluorine is readily displaced from this analog under standard oligonucleotide synthesis deprotection conditions, making its introduction by chemical synthesis challenging43. In contrast 7F-7dA is stable under nucleophilic substitution conditions (Scheme 1), and could be incorporated site specifically via chemical synthesis.…”

2′-Deoxy-2-fluorotubercidin

“…This might be caused by the strong electron-withdrawing effect of the 2-¯uoro atom (pK a < 1.5; Peng et al, 2006). The N3ÐC2 [1.305 (3) A Ê ] and C2ÐN1 [1.315 (3) A Ê ] bond lengths in (III) are shorter than those in (IV) (N3ÐC2 = 1.335 A Ê and C2ÐN1 = 1.333 A Ê ).…”

“…Thus, 2 H -deoxy-2-¯uorotubercidin, (III), was prepared and its activity and base-pairing properties were studied (Peng et al, 2006). Thus, 2 H -deoxy-2-¯uorotubercidin, (III), was prepared and its activity and base-pairing properties were studied (Peng et al, 2006).…”

Analytical consideration of the selectivity of oligonucleotide hybridization

“…The ratio of equilibrium constants of perfect and imperfect complex formation As it can be seen from the expression  recognition. The most promising compounds are PNA [25]; LNA [4], cyclic, cross-linked, and bicyclic oligonucleotides [26,27], oligonucleotides bearing minor groove binders [28], and probes containing analogues of nitrogen bases [30,57]. The selectivity function was shown to reach two upper values, which were determined by the distinctive behavior of this function in two cases: temperature was varied, or it was not.…”