Complexes of Telomeric Oligonucleotide d(TTAGGG)_4 with the New Recombinant Protein Vector PGEk Carrying Nucleic Acids into Proliferating Cells

摘要

A study was made of the complexation of the protein vector PGEk,which transfers nucleic acids into the nuclei of cancer cells,with phosphodiester d(TTAGGG)_4(TMO)and phosphorothioate Sd(TTAGGG)_4(TMS)oligonucleotides,which inhibit telomerase.PGEk(64 amino-acid residues)contains a hydrophobic domain that originates from the human epidermal growth factor(hEGF)and is responsible for the receptor-mediated transfer of PGEk across the cell membrane,and the hydrophilic domain,which is a nuclear localiza-tion signal(NLS)and serves to bind DNA and deliver it to the cell nucleus.Experiments were performed in 0.01-M Na-phosphate and 0.1-M NaCl at 37deg C.An analysis of the circular dichroism(CD)spectra showed that TMO forms an antiparallel G-quadruplex,while TMS occurs in the form of unfolded strands.The number of PGEk molecules adsorbed on oligonucleotides was estimated from the quenching of PGEk fluorescence and the increase in its polarization upon titration with oligonucleotides.Adsorption isotherms were plotted in Scat-chard coordinates.Adsorption of the first two PGEk molecules on TMO and TMS followed a noncooperative mechanism and was characterized by high association constants:K_1(TMO)=(7 +- 1)centre dot 10~7 M~(-1)and K_(1(TMS))=(3 +-0.5)centre dot 10~7 M~(-1).Further adsorption,up to five or six PGEk molecules per TMO molecule,showed high cooper-ation and K_(2(TMO))=(4.0 +- 1.5)centre dot10~6 M~(-1).Unlike TMO,TMS only weakly bound the third PGEk molecule:K_(2(TMS))=(8 +- 2)centre dot 10~5 M~(-1).An analysis of the CD spectra showed that PGEk partly unfolded the G-quadruplex formed by TMO and did not have an effect on the single-stranded structure of TMS.The secondary structure of DNA and the number of protein subunits were established for the biologically active complexes PGEk-TMO and PGEk-TMS,which efficiently pass across the membrane of cancer cells and inhibit their proliferation.