2 ,5 -Phosphodiester

The fundamental problem of oligodeoxyribonucleotide synthesis is the efficient formation of the intemucleotidic phosphodiester bond specifically between C-3 and C-5 positions of two adjacent nucleosides. Any functional group (NH of nucleic base the other OH of deoxy-... 

In the diester method a deoxynucleoside-5 -monophosphate is condensed with the 3 -OH group of a deoxynucleotide to produce a 3, 5 -phosphodiester. This is illustrated by a general method for dinucleotide synthesis developed by H.G. Khorana (K.L. Agarwal, 1976). One N-... 

The ideal phosphorylating reagents for phosphodiester syntheses should meet the following criteria ... 

The same kind of 5 3 phosphodiester units that join the 2 deoxy d ribose units m Figure 28 I are also responsible for connecting nucleosides of d ribose... 

Two new strands form as nucleotides that are complementary to those of the original strands are joined by phosphodiester linkages The sources of the new bases are dATP dGTP dCTP and dTTP already present in the cell... 

The discovery of nbozymes (Section 28 11) in the late 1970s and early 1980s by Sidney Altman of Yale University and Thomas Cech of the University of Colorado placed the RNA World idea on a more solid footing Altman and Cech independently discovered that RNA can catalyze the formation and cleavage of phosphodiester bonds—exactly the kinds of bonds that unite individual ribonucleotides in RNA That plus the recent discovery that ribosomal RNA cat alyzes the addition of ammo acids to the growing peptide chain in protein biosynthesis takes care of the most serious deficiencies in the RNA World model by providing precedents for the catalysis of biologi cal processes by RNA... 

Section 28 6 Many important compounds contain two or more nucleotides joined together by a phosphodiester linkage The best known are those m which the phosphodiester joins the 5 oxygen of one nucleotide to the 3 oxygen of the other... 

Oligonucleotides contain about 50 or fewer nucleotides held together by phosphodiester links polynucleotides can contain thousands of nucleotides... 

Phosphodiester (Section 28 6) Compound of the type shown especially when R and R are D nbose or 2 deoxy D nbose... 

The other important feature of the primary stmcture of RNA is the presence of the 2 -hydroxyl group in ribose. Although this hydroxyl group is never involved in phosphodiester linkages, it does impose restrictions on the heHcal conformations accessible to double-stranded RNA. 

HammerheadRtbozyme. A small RNA molecule that catalyzes cleavage of the phosphodiester backbone of RNA is known as the hammerhead ribozyme. This ribozyme occurs namrally in certain vimses where it facihtates a site-specific self-cleavage at the phosphate and generates a 2 3 -cychc phosphate and a 5 -hydroxyl terminus. The reaction requires a divalent metal ion, such as or, as a cofactor. Whereas the... 

Modification of the Phosphodiester Backbone. Oligonucleotides having modified phosphate backbones have been extensively studied (46). Because altering the backbone makes derivatives generally more resistant to degradation by cellular nucleases, these materials have the potential to be more resilient antisense dmgs. 

Phosphorothioates. All three synthetic approaches appHcable to unmodified oligonucleotides can be adapted for synthesis of phosphorothioates (11) (33,46). If all of the phosphodiester linkages in an oligonucleotide are to be replaced with phosphorothioates, the ff-phosphonate method for coupling, followed by oxidation with Sg in carbon disulfide and triethylamine in the final step, is the most straightforward method. 

The methylphosphonates differ from the phosphodiesters and phosphorothiolates in that the methyl derivatives are uncharged and are thus less water soluble. Moreover, compared to the naturally occurring phosphodiesters, the methylphosphonates form slightly less stable duplexes with complementary DNA and RNA sequences. This effect has been ascribed to the iaevitable chiraUty problem that is, if one isomer biads less well, the overall binding is decreased. Methylphosphonates can enter cell membranes by a passive mechanism and are completely resistant to nucleases. 

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

---