Book of Abstracts: Albany 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
Irina A. Besschetnova1,*
1Engelhardt Institute of Molecular Biology