Internucleotide bond

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

An alternative approach to the synthesis of internucleotide bonds is to use an insoluble condensing agent, such as poly(3,5-diethylstyrene)sulphonyl chloride.116... 

Ribonucleases (RNases) may be defined as phosphodiesterases that attack the internucleotide bonds in ribonucleic acid (RNA) and its products but not those in deoxyribonucleic acid (DNA) or simple phos-phodiesters such as bis-p-nitrophenyl phosphate. 

Ribonuclease T2 is regarded as a nonspecific endoribonuclease [EC 2.7.7.17, ribonucleate nucleotido-2 -transferase (cyclizing)]. It preferentially splits the internucleotide bonds between the 3 -adenylic acid group and the 5 -hydroxyl group of adjacent nucleotides in RNA, with the intermediary formation of adenosine 2, 3 -cyclic phosphate and splits consequently all secondary phosphate ester bonds of other nucleotides in RNA via the nucleotides 2, 3 -cyclic phopshates. 

An elegant study of the kinetics has been performed on single-stranded biosynthetic polymers (58), prepared by the method of Bollum et al. (59). Three polymers were used (1) d([3H]pA)B(pA)r2e, (2) d([3H]pA)5(pA)uo ([2- C]pA) - and (3) d([3H]PT)t(PA)in. The digestion was carried out in the presence of Mg2+ and allowed to proceed in a pH stat until 10% of internucleotide bonds were hydrolyzed. The reaction was stopped by heating on a steam bath. The size of the products in the digestion mixture was determined by means of chromatography on a column of Bio-Gel P-60 previously calibrated with oligomers. 

In Section VI the comparison of the rate of hydrolysis of (dA)n and (dT) was discussed. The only way to account for the contradictory findings in different laboratories is to ascribe the differences to the activating ion used. The evidence exists that during the late phase of the reaction factors other than the Pu-pPy specificity determine the lability of the internucleotide bond. Thus, Potter et al. (42) easily digested d-ApApTp to d-ApA and d-pTp, whereas Khorana (48) observed that d-CpApT was resistant to DNase I. Both compounds had Pu-pPy sequence in the /3-y positions, but differed with respect to 3 -terminal phosphate, which exerts a labilizing influence on the proximal internucleotide bond (see below). 

The facts that basic proteinoids catalyze the synthesis of both peptide and internucleotide bonds and that they do this in suspensions of microspheres, i.e. in the same microlocale has led to the suggestion that microspheres on the primitive Earth were the site of development of the coded genetic mechanism 53). 

It should be noted that the allyl group serves as efFicient protector for internucleotide bonds and nucleoside bases, respectively, in the solution-phase synthesis of nucleic acids. ... 

E. coli can divide every 40 minutes. Thus, its DNA (MW = 2.2x 10 ) can be duplicated in 40 minutes (or less). Calculate (a) the number of internucleotide bonds made per minute, (b) the rate of chromosome duplication in terms of mm/min and /xm/min (assuming only one growing point), and (c) the rate at which the double helix unwinds (turns/min) during duplication. 

The result of this correlation indicates that a sequence of reactions slow-[180]-15b—> [180]-17b—>P180-dinucleotide 32yields the [180]phosphate internucleotide bond of Rp absolute configuration. It seems reasonable to assume that the oxidation of [180]-15 into [180]-17,as well as the condensation of [180]-17 with 3 -0-acetylthymidine 16, both occur with retention of configuration. 

The two sets of 180-labeled monomers [180] -15 and [180] -17 described above and their unlabeled counterparts allowed for the synthesis of medium size oligonucleotides with any combination of internucleotide bonds with respect to... 

However, it is known that the presence of a sulfur atom influences the properties of internucleotide bonds, mostly due to different steric requirements of... 

Picryl chloride eppeoni to he promising fur formation of internucleotide bonds. ... 

Adenosine 5 -phosphorimidazolidate condenses in aqueous solution in the presence of divalent metal ions to form oligo(adenylic acid). Using divalent lead ions, as much as 57% condensation to oligo(A) has been reported studies on the di-and tri-nucleotide with RNase Tg show the internucleotide bonds to be mainly 2 ->5 -linked. The metals most efficient in catalysing condensation occupy an intermediate position in the classification as hard and soft acids and bases, and it is thought that they may co-ordinate to both base and phosphate. 

Many other ribonucleases have been described. They differ with respect to specificity toward various internucleotide bonds, in heat stabilities, and in pH optima. In 1949 Maver and Greco (M8) reported the presence in extracts of spleen of a heat-labile nuclease that had a pH optimum of approximately 5.2 in the presence of magnesium, and 6.6 in the absence of magnesium. That this was a different enzyme entirely from pancreatic RNase was shown most dramatically by Hilmoe and Heppel (H7), who found that this enzyme was able to hydrolyze the limit polynucleotide obtained by exhaustive treatment of RNA with ribonuclease I. The ubiquity of ribonucleases can perhaps best be demonstrated by noting... 

The IDTr group was developed to protect the 5 -OH of deoxyribonucleotides and to increase the rate of internucleotide bond formation through participation of the pendant imidazole group. Rate enhancements of 350 were observed except when (i-Pr)2EtN was added to the reaction mixture, in which case reactions were complete... 

Prepared for use in the phosphoramidite approach, the amidite reagent, (CF3)2CHCH20P(N/Pr)2 is stable to distillation unlike the cyanoethyl version which tends to decompose. It is cleaved rapidly with ammonia from the internucleotidic bonds. ... 

BU4NF, THF, Pyr, H2O, rt, SOmin. These conditions result in the formation of a mixture of fluorophosphate, and phosphate. In the case of oligonucleotides some internucleotide bond cleavage is observed with this reagent. 

Halogen-substituted phenols were originally introduced for phosphate protection to minimize internucleotide bond cleavage during deprotection. ... 

The rate of oligonucleotide synthesis by the triester method using mesitylenesulfonyl chloride was increased 5- to 10-fold when this group was used as a protective group during internucleotide bond formation. It was removed with coned. NH4OH at 60°C for 12 h or by the oximate method. ... 

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