Influence of cationic molecules on the hairpin to duplex equilibria of self-complementary DNA and RNA oligonucleotides

Abstract

A self-complementary nucleotide sequence can form both a unimolecular hairpin and a bimolecular duplex. In this study, the secondary structures of the self-complementary DNA and RNA oligonucleotides with different sequences and lengths were investigated under various solution conditions by gel electrophoresis, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy and a ultraviolet (UV) melting analysis. The DNA sequences tended to adopt a hairpin conformation at low cation concentrations, but a bimolecular duplex was preferentially formed at an elevated cationic strength. On the other hand, fully matched RNA sequences adopted a bimolecular duplex regardless of the cation concentration. The thermal melting experiments indicated a greater change in the melting temperature of the bimolecular duplexes (by approximately 20 degrees C) than that of the hairpin (by approximately 10 degrees C) by increasing the NaCl concentration from 10 mM to 1 M. Hairpin formations were also observed for the palindrome DNA sequences derived from Escherichia coli, but association of the complementary palindrome sequences was observed when spermine, one of the major cationic molecules in a cell, existed at the physiological concentration. The results indicate the role of cations for shifting the structural equilibrium toward a nucleotide assembly and implicate nucleotide structures in cells.

Figure 1

(A) Oligonucleotide sequences and their abbreviations. Underline indicates the base substitutions from d14cg or r14cg. (B–E) Gel electrophoresis at 4°C for 20 μM DNA (B and C) and RNA nucleotides (D and E), prepared in 10 mM (B and D) or 1 M NaCl (C and E). The nucleotides on the marker lanes are the non-self complementary duplex of 5′-ACTGAGGACCTA-3′/5′-TAGGTCCTCAGT-3′ (M_D), the stable tetraloop structure of 5′-GGAGCTTGCTCC-3′ (M_H) (12), and a single strand of 5′-ACTGAGGACCTA-3′ (M_S). D and H on the right side of the gel images indicate the bimolecular duplex and the hairpin, respectively.

Figure 2

Dependence of the fractions of the bimolecular duplex and the hairpin on the NaCl concentration. The gel electrophoresis of (A) d14ta and (B) d12ta and (C) r14cg-caag at 20 μM with an increase in the NaCl concentration; 10, 20, 40, 50, 70, 100, 150, 200, 400 and 1000 mM from left to right. (D) The profiles of the fraction of the bimolecular duplex versus NaCl concentration obtained by gel electrophoresis for d14ta (gray squares), d12ta (white triangles), r14cg-caag (black circles), based on the CD spectra change at 265 nm for r14cg-caag (black triangles), and by gel electrophoresis with the fluorescein-labeled d14ta (gray diamonds).

Figure 3

(A) CD spectra of r14cg-caag at various NaCl concentrations measured at 4°C. The arrows indicate the change in the NaCl concentration from 10 mM to 1 M. (B) Structure of the nitroxide spin-labeled deoxyuridine and the sequences of d14ta and d12ta containing the spin label deoxynucleoside (denoted by S). (C) The EPR spectra of the spin labeled d14ta (upper), d12ta (middle) and a single-strand of 5′-TGAGGSGCTGTTTGTG-3′ as the control (lower) measured in 10 mM NaCl (black) and 1 M NaCl (gray).

Figure 4

(A) Sequences of the DNA nucleotides in addition to those indicated in Figure 1A. Gel electrophoresis at 4°C of (B) 20 μM fluorescein-labeled DNAs at 10 mM NaCl, (C) 20 μM DNAs at 1 M NaCl and (D) 1 μM DNAs at 10 mM NaCl. The marker lanes (M_D, M_H and M_S) are the fluorescein-labeled nucleotides identical to those examined in Figure 1.

Figure 5

(A) Palindrome DNA sequences derived from the ColE1 sequence in E.coli. The nucleotides were labeled either with 6-FAM or by carboxytetramethylrhodamine (TAMRA) at the 5′ end. The terminal non-self complementary sequences are indicated in bold. (B) The PAGE images of the 43mer DNAs stained by GelStar (left), visualized by the fluorescence of 6-FAM (middle), and visualized by the fluorescence of TAMRA (right). The 6-FAM images of (C) 37mer, (D) 31mer, (E) 25mer and (F) 19mer DNAs. The equilibrium was reached at 100 mM NaCl and different spermine concentrations indicated on the top of the gel images (mM) at 37°C. M indicates the 10 bp DNA ladder marker. D and H on the right side of the gel images indicate the bimolecular duplex and the hairpin, respectively.