Thiol-oligonucleotides

This is why the synthesis of thiol oligonucleotides has always been the center of attention for many nucleic acid chemists. This chapter highlights the 3 -thiol modification involving solid supports. 

Fig. 4. Monomaleimido-Nanogold with thiol-oligonucleotide reaction scheme.

Maleimido-peptide and thiol-oligonucleotides are deblocked just before use. 

Figure 2 Chemical structures of the commercially available phosphoramidite and solid support used for the preparation of thiol-oligonucleotides.

N-(4-[ F]fluorobenzyl)-2-bromoacetamide 3/90/20-25 Useful for thiols (oligonucleotides) Dolle etal. (1997) Tavltlan et al. (1998)... 

Luderer F, Walschus U (2005) Immobilization of Oligonucleotides for Biochemical Sensing by Self-Assembled Monolayers Thiol-Organic Bonding on Gold and Silanization on Silica Surfaces. 260 37-56... 

Many of the chemical derivatization methods employed in these strategies involve the use of an activation step that produces a reactive intermediary. The activated species then can be used to couple a molecule containing a nucleophile, such as a primary amine or a thiol group. The following sections describe the chemical modification methods suitable for derivatizing individual nucleic acids as well as oligonucleotide polymers. 

Figure 27.6 The 5 -phosphate group of oligonucleotides may be labeled with cystamine using the EDC/imid-azole reaction. This results in the formation of an amine-terminal spacer containing an internal disulfide group. Reduction of the disulfide provides a route to creating a free thiol for further derivatization.

Figure 27.8 SATA may be used to modify a 5 -amine derivative of an oligonucleotide, forming a protected sulf-hydryl. Deprotection with hydroxylamine results in generation of a free thiol.

Prepare 10-20 pg of a sulfhydryl-containing oligonucleotide in 200 pi of 50 mM sodium phosphate, lOmM EDTA, pH 7.2 (the methods outlined in Section 2.2 of this chapter can be used to form the thiol group). 

Gaur, R.K. (1991) Introduction of 5 -terminal amino and thiol groups into synthetic oligonucleotides. Nucleoside Nucleotides 10, 895-909. 

Ghosh, S.S., Kao, P.M., McCue, A.W., and Chappelle, H.L. (1990) Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes. Bioconjugate Chem. 1, 71-76. 

The microspheres—synthesised via a two-step process (acid-catalysed hydrolysis and condensation of 3-mercaptopropyltrimethoxysilane (MPS) in aqueous solution, followed by condensation catalysed by triethanolamine)—have a narrow size distribution (Figure 5.16) and are considerably more stable than polystyrene divinylbenzene microspheres as shown in phosphoramidite oligonucleotide synthesis by the excellent retention of fluorescence intensity in each of the reagent steps involved in phosphoramidite DNA synthesis (Figure 5.17, in which the organo-silica microsphere free thiol groups are derivatized with ATTO 550 maleimide coupled to the entrapped dye). 

Different types of nucleic acids or their analogues (cDNA, oligonucleotides or peptide nucleic acids), supports (silica, gold, polymeric membranes and gels), surface activation chemistries (organosilanes, thiols) and patterning tools can be used for these purposes and will be described in this review. 

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