Phosphodiesters, Oligonucleotides, and Polynucleotides

RNA ligases catalyze the 3 —> 5 phosphodiester bond formation of RNA molecules with concomitant hydrolysis of ATP to AMP and PP . Initially detected as an activity that catalyzes the circularization of polyribonucleotides with 3 -OH and 5 -P ends (1), the RNA ligase is found in E. coli after infection by T-even (but not T-odd) bacteriophages. The enzyme can carry out intermolecular as well as intramolecular ligation reactions on ssRNA, ssDNA, or a variety of oligonucleotides and polynucleotides having 5 -P and 3 -OH termini. 

A 0.7- to 1.3-ppm downfield shift has been observed for a variety of oligonucleotides and polynucleotides with increasing temperatures (Gorenstein et al., 1976b). llus corresponds to a change from the all g,g phosphodiester conformation in the stacked helical structure (Sundaralingam, 1969 Day et al., 1973) to a mixture of g,g and t,g conformations in the unstacked, random-coil structure. 

Oligonucleotides linear sequences of up to 20 nucleotides, joined by phosphodiester bonds. Position 3 of each nucleotide unit is linked via a phosphate group to position 5 of the next unit. In the terminal units, the respective 3 and 5 positions may be free (i.e.-OH groups) or phosphorylated. O. are named according to chain length, i. e. di-, tri-, tetra-, pentanu-cleotides, etc. Linear sequences of more than 20 nucleotide units are called Polynucleotides (see and compare). 

Like all ribonucleases, pancreatic ribonuclease attacks phosphodiester linkages between the 3 and 5 positions of the RNA nucleotide moieties to yield 2, 3 -cyclic phosphate derivatives. The cyclic phosphates are then further hydrolyzed to yield the 3 -phosphate derivatives. The specificity of pancreatic ribonuclease is such that it restricts its activity to the phosphodiester bonds that involve 3, 5 -linkages between a pyrimidine and any other base. Therefore, the product of the ribonucleic acid digestion with ribonuclease contains 3 -pyrimidine mononucleotides and oligonucleotides with a 3 -cytidylic or uridylic mononucleotide. Taka-diastase (Tl) is a ribonuclease that attacks the polynucleotides to yield 3 guanosine mononucleotides and oligonucleotides with a terminal 3 -guanylic acid. 

Reaction conditions have been described for the synthesis of oligonucleotides by the use of polynucleotide phosphorylase, which uses a dinucleoside (3 -50-nionophosphate as primer and nucleoside 5 -phos-phates. The enzyme also used as primers unnatural phosphodiesters, such as nitrophenylated nucleoside 5 -phosphates. The yields of these reactions, however, are considerably lower as compared to reactions with natural phosphodiesters as primer. The yields of the reaction were found to be relatively insensitive to variations in salt concentration or in pH of the incubation mixtures. Optimal salt conditions, i.e., mM MgCh and 0.4-1,0 MNaCl, are similar to those that are used in oligonucleotide synthesis with normal primers. However, it is advisable that incubation mixtures not exceed volumes of 1 ml. In our experience, it is better to split larger volumes into smaller ones, if large quantities of oligonucleotide are to be synthesized. 

Schaller, H, Weimann, G, Lerch, B, and Khorana, H G (1963) Studies on polynucleotides XXIV. J. Am. Chem. Soc. 85,3821-3827 Schneider, K. C. and Benner, S A (1990) Building blocks for oligonucleotide analogs with dimethylene-sulfide, -sulfoxide, and -sulfone groups replacing phosphodiester linkages. Tetrahedron Lett. 31, 335-338. 

---