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Complexes of telomeric oligonucleotide d(TTAGGG)4 with the new recombinant protein vector PGEk carrying nucleic acids into proliferating cells

  • Structural-Functional Analysis of Biopolymers and Their Complexes
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Abstract

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 localization 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 37°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 Scatchard 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) · 107 M−1 and K 1(TMS) = (3 (± 0.5) · 107 M−1. Further adsorption, up to five or six PGEk molecules per TMO molecule, showed high cooperation and K 2(TMO) = (4.0 ± 1.5) · 106 M−1. Unlike TMO, TMS only weakly bound the third PGEk molecule: K 2(TMS) = (8 ± 2) · 105 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.

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Authors and Affiliations

  1. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia

    I. A. Besschetnova, A. K. Shchyolkina & O. F. Borisova

  2. Bioengineering Center, Russian Academy of Sciences, Moscow, 119312, Russia

    G. E. Pozmogova

  3. Institute of Physico-Chemical Medicine, Ministry of Health and Social Development of the Russian Federation, Moscow, 119992, Russia

    A. N. Chuvilin

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  1. I. A. Besschetnova
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  2. G. E. Pozmogova
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  3. A. N. Chuvilin
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  4. A. K. Shchyolkina
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  5. O. F. Borisova
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Original Russian Text © I.A. Besschetnova, G.E. Pozmogova, A.N. Chuvilin, A.K. Shchyolkina, O.F. Borisova, 2006, published in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 3, pp. 489–496.

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Besschetnova, I.A., Pozmogova, G.E., Chuvilin, A.N. et al. Complexes of telomeric oligonucleotide d(TTAGGG)4 with the new recombinant protein vector PGEk carrying nucleic acids into proliferating cells. Mol Biol 40, 433–439 (2006). https://doi.org/10.1134/S0026893306030101

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