Book of Abstracts: Albany 2005

category image Volume 22
No. 6
June 2005

The Effect of the Secondary and Tertiary Structure of d(TTAGGG)n Telomeric Oligonucleotides on their Binding with a Novel Recombinant Protein PGEk ? Deliver of DNA in Cells

Improving the methods for oligonucleotide and gene delivery into cells is important development of antisense and gene therapy. PGEk (Protein Gen-carrier based on Epidermal Growth Factor) is a new recombinant protein constructed by Pozmogova and co-authors for antisense oligonucleotides and plasmid DNA target delivery into actively proliferating cells due to receptor-mediated endocytosis (1, 2). PGEk cell targeting moiety is composed of EGFh (human epidermal growth factor) and nuclear localization signal (NLS) containing 4 lysine residues on the NH2-end that serve as a DNA-binding moiety. Experiments in vitro have demonstrated that d(TTAGGG)4 oligonucleotide delivery by PGEk into overexpression EGF receptor tumor cells HeLa, MCF-7 and A431 is able to suppress cell growth (IC50 < 500 nM) (1, 2).

In this study we investigated the effect of telomeric DNA structures on PGEk-DNA complex formation. The G-quadruplex structure formed by telomeric sequence d(TTAGGG)4 is known to inhibit telomerase (3) that is upregulated in tumor cells. The binding PGEk to G-quadruplex formed by d(TTAGGG)4 was studied and compared with that to the hairpin-forming d(TTAGGG)3 oligonucleotide and d(GT)12 strand. Samples contained 10mM Na-phosphate buffer, pH 7.5, 0.1 M NaCl, temperature was at 3°_. Formation of the PGEk-DNA complex was detected by independent methods: measurement of the intrinsic tryptophane fluorescence of the protein, circular dichroism (CD), and polarized fluorescence of etidium bromide probe.

Two-stage mode of PGEk complex formation with DNA was detected by analysis of the binding curves. The first three PGEk molecules bound non-cooperatively to the d(TTAGGG)4 G-quadruplex with dissociation constant Kd = 13 ± 2 nM. Subsequent binding of 5-6 PGEk molecules occurred cooperatively with higher dissociation constant Kd = 250 ± 50 nM. CD spectra analysis in the wavelength range 200-320 nm revealed a partial unfolding of G-quadruplex upon binding PGEk molecules. Binding of the first two PGEk molecules to d(TTAGGG)3 and d(GT)12 oligonucleotides (Kd = 25 ± 5 nM) did not depend on their secondary structure, and subsequent binding of up to 3-4 molecules PGEk occurred with a distinct cooperativity (Kd = 400 ± 50 nM).

Thus, we demonstrated a binding preference of PGEk polypeptide vector to the G-quadruplex structure of d(TTAGGG)4 oligonucleotide. We propose the PGEk-DNA complexes as a tool for potential development in gene and antisense therapy.


The study was supported by the RFBR grant N 04-04-49618 and N 05-04-48909.

References and Footnotes
  1. Pozmogova G. E., Chuvilin A. N., Posypanova G. A., Shulga A. A., Ermolyuk Ya. S., Kireeva N. N., Kirpichnikov M. P., and Skryabin K. S. New EGF-based Peptide Vectors for Antisense Oligonucleotides and Plasmid DNA Target Delivery into Actively Proliferating Cells. Abstr. Int. Conf. ?RNA as Terapeutic and Genomics Target?. Novosibirsk. pp. 83 (2001).
  2. Pozmogova G. E. et al. patent N 2003125289/13(027073). Russia (2003).
  3. Zahler A. M., Williamson T. R., Cech T. R., and Prescott D. M. Inhibition of Telomerase by G-quartet Structures. Nature 350, 718-720 (1991).

Irina A. Besschetnova1,*
Galina E. Pozmogova2
Anna K. Shchyolkina1
Olga F. Borisova1

1Engelhardt Institute of Molecular Biology
Russian Academy of Sciences
119991 Moscow, Russia
2Center ?Bioengineering?
Russian Academy of Sciences
Moscow, Russia

*Phone: 7095-1359789
Fax: 7095-1351405
Email: borisova@eimb.ru