5 -Phosphate group of oligonucleotides

Figure 1.112 Phosphate groups can be modified with adipic acid dihydrazide in the presence of a carbodi-imide to produce hydrazide derivatives. This is a common modification route for the 5 -phosphate group of oligonucleotides.

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.

Add to the tube 7.5 pi of RNA or DNA containing a 5 -phosphate group. The concentration of the oligonucleotide should be 7.5-15 nmol or total of about 57-115.5 pg. Also, immediately add 5 pi of 0.25 M adipic acid dihydrazide or carbohydrazide dissolved in 0.1 M imidazole, pH 6.0. Because EDC is labile in aqueous solutions, the addition of the oligo and fezs-hydrazide/imidazole solutions should occur quickly. 

Sulfhydryl groups also can be added to 5 -phosphate end of DNA probes (Chapter 27, Section 2.2). Biotinylation at these sites avoids disruption of base pairing with complementary DNA targets, since the point of modification is restricted to a single end position on the oligonucleotide. 

Figure 27.5 Oligonucleotides containing a 5 -phosphate group can be reacted with EDC in the presence of imidazole to form an active phosphorimidazolide intermediate. This derivative is highly reactive with amine nucleophiles, forming a phosphoramidate linkage. Diamines reacted with the phosphorimidazolide result in amine-terminal spacers that can be modified with detectable components.

This enzyme [EC 3.1.26.6], also known as endoribo-nuclease IV and poly(A)-specific ribonuclease, catalyzes the endonucleolytic cleavage of poly(A) to fragments terminated by 3 -hydroxyl and 5 -phosphate groups. Oligonucleotides are formed with an average chain length of ten. 

Hiramaru et al. 123, 124) isolated four RNases and two nucleases from a slime mold, Physarum polycephalum. One (RNase PP ) of the RNases was found to be a novel enzyme. It hydrolyzes RNA in an endonucleolytic way, producing pA, pG, and oligonucleotides bearing 5 -phosphates group with a high preference for purine with regard to 3 -terminal nucleotides. 

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