Induction of parallel human telomeric G-quadruplex structures by Sr2+

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Abstract

Human telomeric DNA forms G-quadruplex secondary structures, which can inhibit telomerase activity and are targets for anti-cancer drugs. Here we show that Sr2+ can induce human telomeric DNA to form both inter- and intramolecular structures having characteristics consistent with G-quadruplexes. Unlike Na+ or K+, Sr2+ facilitated intermolecular structure formation for oligonucleotides with 2 to 5 5′-d(TTAGGG)-3′ repeats. Longer 5′-d(TTAGGG)-3′ oligonucleotides formed exclusively intramolecular structures. Altering the 5′-d(TTAGGG)-3′ to 5′-d(TTAGAG)-3′ in the 1st, 3rd, or 4th repeats of 5′-d(TTAGGG)4-3′ stabilized the formation of intermolecular structures. However, a more compact, intramolecular structure was still observed when the 2nd repeat was altered. Circular dichroism spectroscopy results suggest that the structures were parallel-stranded, distinguishing them from similar DNA sequences in Na+ and K+. This study shows that Sr2+, promotes parallel-stranded, inter- and intramolecular G-quadruplexes that can serve as models to study DNA substrate recognition by telomerase.

Section snippets

Materials and methods

Telomeric oligonucleotides. HPLC purified oligonucleotides composed of normal or altered human telomeric repeats were ordered from Sigma Genosys. Telomeric oligonucleotides (Tn) consisted of n = 2–9, 5′-dTTAGGG-3′ repeats, while Tn-all had n∗ = 2–9, 5′-dTTAGAG-3′ repeats. Tn-X oligonucleotides had a single or multiple 5′-dTTAGGG-3′ repeat(s) converted to 5′-dTTAGAG-3′ in the 5′ → 3′ direction (X).

Native polyacrylamide gel electrophoresis. Oligonucleotides (2 μM) prepared in SrCl2 (0 or 100 mM), and TE

Results

To investigate the role of Sr2+ in the formation of G-quadruplexes of different DNA length and sequence, oligonucleotides T2–T9 denoting the number of 5′-d(TTAGGG)-3′ repeats ranging from 2 to 9 were synthesized. Furthermore, many of the oligonucleotides had at least one of the 5′-d(TTAGGG)-3′ repeats altered to 5′-d(TTAGAG)-3′ to study the role of the middle guanine in formation of these DNA secondary structures.

Discussion

The results from this study emphasize the diversity of G-quadruplex structures formed by human telomeric DNA. Although Na+ typically stabilizes anti-parallel structures and K+ stabilizes mixed parallel/anti-parallel structures for sequences similar to our T4 and T8, Sr2+ appears to stabilize exclusively parallel-stranded structures. This has also been shown for the sequence 5′-d(G4T4G4T4G4T4G4)-3′ in the presence 2 mM Sr2+[14] and 5′-d(G4T4G4)-3′ in Ca2+[23]. However, Sr2+ does not exclusively

Acknowledgments

We thank Dr. Thomas K. Harris for the use of his UV–vis spectrophotometer and Dr. James D. Potter and Dr. Audrey N. Chang for the use of their spectropolarimeter.

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