2-aminopurine as a probe for quadruplex loop structures

Abstract

Fluorescent reporter groups have served for many years as sensitive probes of macromolecular structure. Such probes can be especially useful in comparative studies such as detection of conformational changes and discrimination among structural models. Spectroscopic methods such as fluorescence are attractive because they are rapid, require small amounts of material, are nondestructive, can be carried out with commonly available equipment, and are relatively inexpensive. In addition, there is a rich library of theoretical and practical materials available to aid in data interpretation.The intrinsic fluorescence of most nucleic acids is too low to be useful in structural studies. Thus, it is necessary to incorporate a suitable reporter group to utilize fluorescence methods involving polynucleotide structure. A highly fluorescent adenine analog, 2-aminopurine, has long served in this capacity. The present article describes our use of 2-aminopurine as a probe of loop structures in quadruplex DNA. In particular, we show how knowledge of the relative intensity of 2-aminopurine emission as well as its sensitivity to exogenous quenching molecules such as acrylamide can aid in comparing crystal and solution structures of an oligonucleotide model of the human telomere and in discrimination among models containing tandem repeats of the telomeric quadruplex.

Fig. 1

G-quartet showing H-bonding pattern and coordinated sodium ion in center. The figure shows a single G-quartet and is based on the coordinates of HuTel22 in PDB file 143D (1). The hydrogen bonding pattern is shown by the thin lines connecting the N and O atoms. The D-ribosyl phosphate moieties are shown on the periphery of the

Fig. 2

Schematic diagram of potential folding topographies of unimolecular quadruplexes. The Na⁺ form of HuTel22 has been shown to exist in solution mainly in the form of an antiparallel basket (1). In the presence of K⁺, HuTel22 in crystals is an all-parallel “propeller” structure (23). In K⁺ solution, NMR and other physical studies indicate that HuTel22 is present as a mixture of different conformers (4,8). The circle represents the 5′ end of the oligonucleotide.

Fig. 3

Structures of adenine and 2-aminopurine

Fig. 4

Emission spectra (panels A and C) and fluorescence quenching curves (panels B and D) for HuTel22 measured in the presence of Na⁺ or K⁺ at 5 °C. The lines were drawn in the Stern-Volmer plots using the optimized parameters K_SV,1, K_SV,2 and f₁ given in Table 1 and determined by non-linear least fitting of the quenching data to Eq. 4 as described in the text. The data are re-plotted from reference (8).