Remarkably Stable Parallel-Stranded Oligonucleotides Containing 5-Methylisocytosine and Isoguanine

Abstract

The polymorphic nature of double-stranded DNA is well established. 1 A common feature of three major families of A-, B-, and Z-DNA duplexes is the antiparallel orientation of the constituent strands. Parallel-stranded DNA (ps-DNA) is a new family of DNA conformations experimentally confirmed in 1988, in which complementary strands have the same 5′-3′ orientation and form reverse Watson-Crick A-T base pairs. 2 Numerous thermodynamic studies revealed that the parallelstranded duplexes melt with considerably lower transition temperatures than the corresponding antiparallel duplexes. 2 Thus, 3′-p-3′-or 5′-p-5′-linked hairpins or relatively long oligomers such as 25-mers have been employed to investigate the thermodynamic behavior and reactivities of ps-DNA. 3 We reasoned that incorporation of 2′-deoxy-5-methylisocytosine (iC) or 2′-deoxyisoguanine (iG) as a new artificial base would stabilize parallel-stranded duplexes by forming three hydrogen bonds with guanine and cytosine, respectively. 4 We now report herein that iC-and iG-containing oligomers can form a parallelstranded duplex of a comparable stability with a normal antiparallel duplex even at a decamer level.PM3 semiempirical calculations in the gas phase suggested that both iC-G (-12.0 kcal) and iG-C (-13.7 kcal) base pairs are more stable than a reverse Watson-Crick A-T base pair (-4.6 kcal) and should possess stability comparable with a Watson-Crick G-C base pair (-11.9 kcal) ( Figure 1). 5 Thus, iG-and iC-containing deoxydecanucleotides (ODN I-III) were synthesized according to the standard -cyanoethyl phosphoramidite chemistry on a DNA synthesizer (Table 1). 6 The -cyanoethyl phosphoramidites of protected iC and iG were prepared according to the reported procedure. 7 After standard deprotection with concentrated ammonia, the oligomers were purified by reverse phase HPLC. Concentration and composition of oligomers were determined by complete digestion of oligomers with snake venom phosphodiesterase and bacterial alkaline phosphatase to mononucleosides.In order to investigate duplex formation, UV spectra of the mixture of ODN I and 5′-d(ACGTGCCTGA)-3′, which can form full-matched base pairs when aligned in a parallel orientation, were examined at various temperatures. As shown in Figure 2a, the mixture exhibited a thermally induced hyperchromicity. The 1:1 stoichiometry for complex formation of each strand was confirmed by UV mixing curve experiments. 8 Figure 2b shows a thermal denaturation profile of ODN I and 5′-d(ACGTGCCTGA)-3′ (p-1) together with p-2 and p-3, showing a cooperative UV melting behavior from base-stacked ps duplex to a single strand. A further indication for the ps-DNA duplex was obtained from CD spectrum of 1:1 mixture of ODN I and 5′-d(ACGTGCCTGA)-3′ as shown in Figure 2c. In accord with the previous observation, 2,3 the CD of p-1 showed a typical negative maximum dichroism near 260 nm, very similar to that observed for antiparallel duplexex.In order to investigate the recognition ability of iC-and iGcontaining oligomers toward single...

Hole delocalization over adenine tracts in single stranded DNA oligonucleotides

“…22,23 Competition between charge localization and delocalization is of paramount importance in all the fields of chemistry, from molecular reactivity to material properties, and it is of particular significance also for charge transport in DNA, especially because the possible establishment of delocalized hole domains can significantly affect the hole site energies and in turn the hole transfer rates. [33][34][35][36][37][38][39][40][41] By contrast, ESR spectra of short oligonucleotides containing three consecutive Gs do not show any evidence of charge delocalization, 42 and similar conclusions also emerge from several computational studies. 32 Evidence pointing toward the establishment of delocalized hole sites has also been provided by Saito et al, who have shown that oxidative damage to DNA preferentially occurs at sites consisting of two or three consecutive guanines (G).…”

Solvent Effects on Ionization Potentials of Guanine Runs and Chemically Modified Guanine in Duplex DNA: Effect of Electrostatic Interaction and Its Reduction due to Solvent

“…The IPs (corresponding to the free energy of the hole) obtained by the ab initio Hartree−Fock (HF) molecular orbital (MO) calculations 19,20 are smaller for the longer guanine run. These calculations were performed for the DNA oligomers without a backbone in the gas phase.…”

State transitions by molecules

“…These oligomers, except for the iG/iC-containing oligomers, were commercially synthesized by Amersham Pharmacia biotech Co., Ltd. (Tokyo). Oligomers iC1, iC2, RiG1, and RiG2 were chemically synthesized (Sugiyama et al, 1996). Two successive transitions were performed in a reaction mixture containing oligomer Tran1 (3 mM), 2 mM MgCl 2 , dATP, dGTP, and dCTP (250 mM each), 2 units of AmpliTaq DNA polymerase (Perkin-Elmer), and the PCR buffer provided by the supplier.…”