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DNA modification of live cell surface.

Borisenko GG, Zaitseva MA, Chuvilin AN, Pozmogova GE - Nucleic Acids Res. (2009)

Bottom Line: By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface.All procedures can be completed within minutes and do not alter cell viability.Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Physical-Chemical Medicine, Russian Academy of Science, Moscow 119312, Russia. grigoryb@yahoo.com

ABSTRACT
We report a novel approach for the attachment of DNA fragments to the surface of live cells. By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface. Method permits to control amount of immobilized DNA on the cell surface. All procedures can be completed within minutes and do not alter cell viability. Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion. Thus, this method allows rapid and simple DNA multicoding of the cell surface and, therefore, opens new opportunities in manipulating with cell-cell interactions.

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Scheme of synthesis and HPLC analysis of fatty acid oligonucleotide conjugates. Fatty acid oligonucleotide conjugate (FT18Ste) was prepared via condensation reaction of FAM-labeled oligomer (FT18N) with stearic acid N-oxysuccinimide. Products were analyzed by using HPLC system equipped with a fluorescent detector (excitation and emission wavelengths were 470 and 520 nm, respectively).
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Figure 2: Scheme of synthesis and HPLC analysis of fatty acid oligonucleotide conjugates. Fatty acid oligonucleotide conjugate (FT18Ste) was prepared via condensation reaction of FAM-labeled oligomer (FT18N) with stearic acid N-oxysuccinimide. Products were analyzed by using HPLC system equipped with a fluorescent detector (excitation and emission wavelengths were 470 and 520 nm, respectively).

Mentions: To attach oligonucleotides to the surface of live cells we constructed amphiphile conjugates of oligonucleotide and fatty acid. The hydrophobic moiety was aimed to anchor an entire molecule at the PM by incorporating into the outer leaflet (Figure 1). Oligonucleotides were synthesized and purified as 3′-aminoalkyl phosphodiester derivatives with a final purity of ≥97% according to HPLC assay. The following condensation reaction of oligonucleotide with N-oxysuccinimide derivative of a fatty acid yielded 50–66% of the conjugate as detected by reverse-phase HPLC (Figure 2).Figure 1.


DNA modification of live cell surface.

Borisenko GG, Zaitseva MA, Chuvilin AN, Pozmogova GE - Nucleic Acids Res. (2009)

Scheme of synthesis and HPLC analysis of fatty acid oligonucleotide conjugates. Fatty acid oligonucleotide conjugate (FT18Ste) was prepared via condensation reaction of FAM-labeled oligomer (FT18N) with stearic acid N-oxysuccinimide. Products were analyzed by using HPLC system equipped with a fluorescent detector (excitation and emission wavelengths were 470 and 520 nm, respectively).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651772&req=5

Figure 2: Scheme of synthesis and HPLC analysis of fatty acid oligonucleotide conjugates. Fatty acid oligonucleotide conjugate (FT18Ste) was prepared via condensation reaction of FAM-labeled oligomer (FT18N) with stearic acid N-oxysuccinimide. Products were analyzed by using HPLC system equipped with a fluorescent detector (excitation and emission wavelengths were 470 and 520 nm, respectively).
Mentions: To attach oligonucleotides to the surface of live cells we constructed amphiphile conjugates of oligonucleotide and fatty acid. The hydrophobic moiety was aimed to anchor an entire molecule at the PM by incorporating into the outer leaflet (Figure 1). Oligonucleotides were synthesized and purified as 3′-aminoalkyl phosphodiester derivatives with a final purity of ≥97% according to HPLC assay. The following condensation reaction of oligonucleotide with N-oxysuccinimide derivative of a fatty acid yielded 50–66% of the conjugate as detected by reverse-phase HPLC (Figure 2).Figure 1.

Bottom Line: By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface.All procedures can be completed within minutes and do not alter cell viability.Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion.

View Article: PubMed Central - PubMed

Affiliation: Research Institute of Physical-Chemical Medicine, Russian Academy of Science, Moscow 119312, Russia. grigoryb@yahoo.com

ABSTRACT
We report a novel approach for the attachment of DNA fragments to the surface of live cells. By using fluorescence microscopy and flow cytometry we demonstrated that our synthetic conjugates of fatty acid with oligonucleotides can be incorporated in plasma membrane and then hybridized with complementary sequences at the cell surface. Method permits to control amount of immobilized DNA on the cell surface. All procedures can be completed within minutes and do not alter cell viability. Using this approach we tethered floating myeloid HL-60 cells to adherent A431 epitheliocytes in a sequence specific fashion. Thus, this method allows rapid and simple DNA multicoding of the cell surface and, therefore, opens new opportunities in manipulating with cell-cell interactions.

Show MeSH
Related in: MedlinePlus