Inosine — Adenosine base pairing in d-GGTACIAGTACC: Duplex and hairpin with a two-base loop

Abstract

Adstract. An oligonucleotide dodecamer d-GGTAClAGTACC containing two inosine-adenosine mismatched base-pairs has been studied by ~H and 3~p NMR spectroscopy. Unique assignments of ~H and ~rp spins have been achieved by using a recently proposed two-dimensional heteronuclear long range correlation (2D-HELCO) teehnique. The 2D nuclear Overhauser enhancement speclroscopy (NOESY) speclrum recorded in a mixed solvent of 90 % 1-I20 + 10 % 2H20 has been used to assign all the exchangeable imino and amino proton resonanees. NMR results indieate that at concentmtions above 5 mM, the molecule adopts a duplex structure where the inosine imino protons are hydrogen bonded. Crossstrand NOEs between the imino proton of 16 and H2 proton of A7 and that between I4_2 of 16 and H2 of A7 provide evidence for inosine-adenosine base-pairing, with both bases 16 and A7 in anti conformation. The observed NOE eonnectivifles dl, a'2 and d3 from almost the middle of the molecule to the two ends are consistent with a ¡ handed B-DNA conformation. However, the molecule adopts a more extended conformation in the mismatch regiun to aceomodate the bulky purine-purine (lA) base pairs. NMR experiments at lower concentrations indicate the coexistente of a monomeric hairpin with a two base loop, eonsisting of 16 and A7 units, in equilibrium with the duplex. 2D-ROESY expe¡ at such a concenlration shows that the conformation of the stem of the hairpin does not deviate much fi'om the corresponding region of the duplex and four nucleotides C5, I6, A7 and G8 undergo significant structural changes during the formation of hairpin loop.

31P–1H correlation and resonance assignments along the DNA backbone: three‐dimensional implementation of heteronuclear long‐range correlation experiment

“…Further, several H4 0 i -H5 0 /H5 00 i TOCSY peaks were also seen in the proton subspectra, which helped in the assignment of some H5 0 /H5 00 protons. As mentioned earlier, Kellog and Schweitzer 30 correlated 31 P with 1 H in DNA by 31 P-1 H cross polarization. Such cross polarization was also adopted for a 3D P-H-H experiment, which gives proton subspectra similar to those discussed in this paper.…”

“…Our experience shows that this procedure is especially useful in situations where the structures of oligomers deviate significantly from the canonical B-DNA. 31 This is primarily because in such situations the chemical shifts of individual 1 H and 31 P spins along the backbone are more dispersed compared with the situation for a canonical B-DNA. We have also used this procedure for complete sequence-specific resonance assignment of 1 H and 31 P spins in situations where the oligomer co-exists in a duplex and hairpin conformation, and the spectra are more complicated owing to the increase in the number of resonances.…”

“…TOCSY spectrum of IA67, recorded on a Bruker AMX 500 spectrometer. The solid lines show sequential connectivities used for the sequence-specific resonance assignment of31 P and 1 H spins along the DNA backbone (see text). Experimental details: 5 mM oligonucleotide solution in 99.9% 2 H 2 O, 40 mM phosphate buffer…”

Evidence for A+(anti)–G(syn) mismatched base–pairing in d–GGTAAGCGTACC

“…Through the use of UV, IR, NMR, gel electrophoresis, and equilibrium ultracentrifugation [5][6][7][8][9] techniques, it has been established that IA67 exists predominantly as a monomeric hairpin in dilute solutions, which is in equilibrium with a duplex of twice the molecular weight. The loop size was shown to be two l\3r the hairpin.…”

“…The loop size was shown to be two l\3r the hairpin. Higher concentration shifts the equilibrium from hairpin to duplex [8,9]. The IA mispair in the duplex adopts I(anti) A(anti) conformation [8].…”