Phosphate group

This same type of modification strategy also can be used to create highly reactive groups from functionalities of rather low reactivity. For instance, carbohydrate chains on glycoproteins can be modified with sodium periodate to transform their rather unreactive hydroxyl groups into highly reactive aldehydes. Similarly, cystine or disulfide residues in proteins can be selectively reduced to form active sulfhydryls, or 5 -phosphate groups of DNA can be transformed to yield modifiable amines. 

DNA or RNA also may be modified with cystamine at the 5 -phosphate group using a carbodiimide reaction. See Chapter 27, Section 2.2 for a complete discussion of the labeling protocol. 

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.

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. 

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.

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.

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. 

The deoxyribonucleotides in the DNA polymer are connected by phosphodi-ester bonds between the 5 -phosphate group attached to one deoxyribose sugar and the 3 -hydroxyl group of the next sugar. 

The 2,3-dideoxyglyc-2-enose also appears to participate in the formation of 5-oxocyclopent-l-enyl phosphate (86) found among the alkaline degradation products of apurinic and apyrimidinic acids.175 The 2,3-dideoxypent-2-enose 5-phosphate (83), formed by -elimination either from apurinic or from apyrimidinic acid, readily isomer-izes to 84, a form in which it can eliminate the 5-phosphate group or, by further rearrangement of the double bonds, cyclize to 85. The fate of 87, also an expected intermediate in the alkaline degradation of 2-deoxy-D-eryf/iro-pentose, is unknown, but it would be expected to cyclize to form 1,3-cyclopentanedione. 

The successive nucleotides of both DNA and RNA are covalently linked through phosphate-group bridges, in which the 5 -phosphate group of one nucleotide unit is... 

The final phosphodiester linkage between the 5-phosphate group on the DNA chain synthesized by DNA polymerase III and the 3 -hydnoxyl group on the chain made by DNA polymerase I is catalyzed... 

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