Oligonucleotides 2 -modified

1 Oligonucleotides containing modified phoqibodiester linkages - Activity in 

The 3 phosphorothioate residues, introduced by the phosphoramidite method, were shown to significantly protect the oligomer from the action of nucleases. This hybrid, in which the ribonucleotide portion is complementary to the leader sequence of phage fl coat protein mRNA, was used to study the formation of the initiation complex in prokaryotic translation. 

Oligonucleoside phosphorodithioates containing all four nucleobases and up to twenty 

Methylphosphonate linkages have been incorporated into DNA enzymatically using the 

The mechanisms by which oligonucleotides cross biological membranes have been investi- 

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Fig. 14. Nonphosphate backbone-modified oligonucleotides where N is a heterocycHc base and R is an alkyl group. See text.

Modified oligonucleotides can be used to cross-link DNA sequences via a reactive group tethered to an oligonucleotide. When irradiated with uv light, psoralens (31) reacts with thymine bases, and the reaction yields a cross-link if the thymine residues are adjacent to each other on opposite strands. Psoralen linked to oligonucleotides have been shown to induce site-specific cross-links in vitro (51). 

Monia BP, Lesnik EA, Gonzalez C, Lima WF, McGee D, Guinosso CJ, Kawasaki AM, Cook PD, Freier SM (1993) Evaluation of 2 -modified oligonucleotides containing 2 -deoxy gaps as antisense inhibitors of gene expression, J Biol Chem 268 14514-14522... 

A variety of methods for modified oligonucleotide preparations have been reported including (1) the C5 and C4 positions for the pyramidines, (2) the exocyclic amino groups and the C8 positions of the purines, and (3) the 2 -hydroxyl group of the carbohydrate residue. Every method should be important as the basis for new immobilization chemistry, however, we especially focused our attention on the use of oligodeoxyribonucleotide phos-phorothioates (Fig. 3). 

Fig. 45. (a) Introduction of M(phen)31 complex into DNA oligomers, (b). Steady-state emission spectra of modified oligonucleotides in 0.01 m sodium phosphate buffer, pH 7.0, 0.1 NaCl. Top Ru(phen)3+-modified 20-mer duplex (solid line), a 1 1 mixture of non-comple-mentary Ru(phen)3+- and Os(phen)g+-modified 20-mers (- -) and a 20-mer duplex containing Ru(phen)g+ and Ps(phen)3+groups on different strands separated by one base pair (---). Bottom 20-mer duplex with 5 -terminal Rufphen) (solid line), and Ru(phen)jf/Os(phen)3 -containing analog (---). Reproduced with permission from Ref. (157). Copyright 1998, American Chemical Society. 

Alguero, B. Lopez de la Osa, J. Gonzalez, C. Pedroso, E. Marchan, V. Grandas, A. Selective platination of modified oligonucleotides and duplex cross-links. Angew. Chem. Int. Ed. 2006, 45, 8194-8197. 

Other problems arise when modified oligonucleotides are synthesized. Oligonucleotides are most commonly synthesized today for pharmaceutical purposes in the form of phosphorothioates (PS), in which sulfurization of the phosphodiester bond has taken place (Figure 1). 

Fluorophosphates (phosphorfluoridates), which are important for backbone modified oligonucleotides in molecular biology, are generally synthesized from phosphoric imi-dazolides or triazolides and acylfluorides. In these reactions, for example, the azole group (of the phosphoric or phosphinic imidazolide) is substituted by a fluorine atom, forming an acylazole compound as by-product.[202],[2033... 

The syntheses, photophysical and electrochemical properties of [Ir(ppy)2(phen-NS-5)]PF6, (181), [Ir(ppy)2(phen-NHCOCH2I-5)]PF6, (182), and [Ir(ppy)2(phen-NH2-5)]PF6 are reported.342 Complexes (181) and (182) have been used to label amine- and sulfhydryl-modified oligonucleotides and human serum albumin to give luminescent bioconjugates. 

DNA and RNA quantification, SNP typing, hybridization, and structural alteration have been widely carried out by modified oligonucleotides possessing pyrene derivatives [104-113]. As is known, pyrene-1-carboxaldehyde fluorescence is considerably dependent on solvent polarity [114], being strong in methanol but insignificant in nonpolar solvents [115]. Owing to this property, Tanaka and collaborators developed a pyrenecarboxamide-tethered modified DNA base, PyU 46, and applied it to SNP discrimination in DNA [116-120],... 

Figure 1.72 Cystamine may be used to label phosphate groups, such as at the 5 -end of oligonucleotides, via a carbodiimide reaction using EDC. The resultant phosphoramidate linkage is a common way to modify oligonucleotides at the 5 -end.

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

Dissolve the amine-modified oligonucleotide to be thiolated in 250pi of 50mM sodium phosphate, pH 7.5. 

Dissolve a 5 -sulfhydryl-modified oligonucleotide in water or lOmM EDTA at a concentration of 0.05-25 pg/pi. Calculate the total nmoles of oligo present based upon its molecular weight. 

Figure 27.15 The homobifunctional crosslinker DSS may be used to conjugate an enzyme to a 5 -diamine-modified oligonucleotide. The NHS ester groups on DSS react with the amines to form amide bonds.

Prepare an amine-modified oligonucleotide according to any of the protocols discussed in Section 2.1 (this chapter). Dissolve or buffer-exchange the oligo into 0.1M sodium borate, 2mM EDTA, pH 8.25, at a concentration of 9 nmol (2.0 A2gonm units) in 15 pi. 

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