[Complexes of telomeric oligonucleotide d(TTAGGG)4 with the new recombinant protein PGEk--nucleic acid carrier into proliferating cells].
The complexation of the new protein vector PGEk--a carrier of nucleic acids into proliferating cells with phosphodiester d(TTAGGG)4 (TMO) and phosphorothioate Sd(TTAGGG)4 (TMS) telomerase inhibitor oligonucleotides was studied. PGEk molecule, consisting of 64 amino acids, is comprising the sequence of the human epidermal growth factor EGFh which is hydrophobic cell targeting moiety serving for receptor-mediated endocytosis and an NLS (nuclear localization signal) which is hydrophilic serving as a DNA-binding and selective nuclear import moiety. Experiments were carried out in 0.01 M Na-phosphate buffer contained 0.1 M NaCl, pH 7.8 at 37 degrees C. CD spectral analysis revealed that TMO molecules folded back into intramolecular antiparallel G-quadruplex while TMS molecules were represented as unstructured thread. The number of adsorbed PGEk molecules were estimated using PGEk intrinsic fluorescence decrease and fluorescence polarization increase of PGEk under oligonucleotide titration. Adsorption isotherms were plotted in Scatchard coordinates. We have shown that adsorption of the first two PGEk molecules on TMO and TMS occurs noncooperatively with the high association constants K1(TMO) = (7 +/- 1) x 10(7) M(-1) and K1(TMS) = (3 +/- 0.5) x 10(7) M(-1), respectively. Further adsorption up to 5-6 PGEk molecules on TMO occurrs cooperatively with still high association constant K2(TMO) = (4.0 +/- 1.5) x 10(6) M(-1). TMS oligonucleotide binds the third PGEk molecule rather weakly, K2(TMS) = (8 +/- 2) x 10(5) M(-1). CD spectral analysis revealed that G-quadruplex structure formed by TMO have undergone a partial unfolding by binding of PGEk molecules while single-stranded structure formed by TMS was not affected by binding PGEk. Thus, the tertiary structure of DNA and the number of adsorbed PGEk molecules formed biologically active compounds PGEk: TMO and PGEk: TMS were defined, which are able to penetrate through the membrane of proliferating cells and to suppress their growth.
Besschetnova
I A
IA
Pozmogova
G E
GE
Chuvilin
A N
AN
Shchelkina
A K
AK
Borisova
O F
OF
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002467:Cell Nucleus; D049109:Cell Proliferation; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004815:Epidermal Growth Factor / Q000737:chemistry*; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D019098:Telomerase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D016615:Telomere / Q000737:chemistry*
D004791:Enzyme Inhibitors; D009838:Oligodeoxyribonucleotides; D004815:Epidermal Growth Factor; D019098:Telomerase