Activation of ODNs

The amino-functionalized ODN is first activated to react with amino groups on latex amine surface. The PDC is first chemically grafted to the amino-linker borne on ODN 5' extremity; the remaining isothiocyanate group should react with amino groups at the latex surface (Fig. 18) [17]. Capillary gel electrophore-sis technique is mainly used to separate ODNs differing by only one nucleotide in their length. Electrophoregrams of activated dT35 ODNs showed a large shift on its retention time compared to the nonactivated one, which could not solely be explained by one PDC addition.

MALDI-TOF mass spectrometry confirmed this substantial increase in molecular weight. An average of four PDC/ODN chains were found, indicating that amines located at 3' position (N-3) on the thymine cyclic moiety react with PDC. The chain distribution for activated ODNs was also broader than that of nonactivated ones, reflecting that the number of PDC reactions on nucleic bases varies significantly on the same ODN sample.

Enzymatic hydrolysis of the ODNs followed by reversed-phase HPLC analysis provided direct proof of base activation, as showed in Fig. 19. Apart from dT and dC (used as a marker) peaks, two new peaks showed up at high retention times belonging to more hydrophobic molecules. One was attributed to the 5' amino-link arm substituted with PDC (confirmed by coinjection), whereas the second peak was assigned to the thymine nucleoside bearing one PDC molecule. Peak integration and comparison with their expected values (according to MALDI-TOF molar mass) confirmed these peak allocations. The reaction of the amine groups of the aromatic bases with the activating agent (PDC) was then clearly evidenced.

Latex Particle

FIG. 18 Methodology of covalent immobilization of ODNs onto aminated latex particles. Step 1: activation reaction of the ODNs by PDC. Step 2: grafting of the activated ODNs onto aminated latex particles.

Latex Particle

FIG. 18 Methodology of covalent immobilization of ODNs onto aminated latex particles. Step 1: activation reaction of the ODNs by PDC. Step 2: grafting of the activated ODNs onto aminated latex particles.

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