Nucleic acid reaction conditions

Amides, like esters, are abundant in all living organisms—proteins, nucleic acids, and many pharmaceuticals have amide functional groups. The reason for this abundance of amides, of course, is that they are stable to the conditions found in living organisms. Amides are the least reactive of the common acid derivatives and undergo relatively few nucleophilic acyl substitution reactions. 

Nucleic acids are anionic under the neutral conditions. Thus, the syntheses of model compounds of the opposite charge are interesting for the discussion of electrostatic contributions in specific interactions of nucleic acids. We have tried to synthesize cationic models by the Menschutkin reaction of poly-4-vinylpyridine with 9-(2-chlo-roethyl)adenine, l-(2-chloroethyl)thymine, and 7-(2-chloroethyl)theophylline15,16 The obtained polymers are poly l-[2-(adenin-9-yl)ethyl]-4-pyridinioethylene chloride 7(APVP), poly l-[2-(thymin-l-yl)ethyl]-4-pyridinioethylene chloride 8 (TPVP), and poly l-[2-(theophyllin-7-yl)ethyl]-4-pyridiniothylene chloride 9 (THPVP), respectively. 

Seasonal variations in the metabolic fate of adenine nucleotides prelabelled with [8—1-4C] adenine were examined in leaf disks prepared at 1-month intervals, over the course of 1 year, from the shoots of tea plants (Camellia sinensis L. cv. Yabukita) which were growing under natural field conditions by Fujimori et al.33 Incorporation of radioactivity into nucleic acids and catabolites of purine nucleotides was found throughout the experimental period, but incorporation into theobromine and caffeine was found only in the young leaves harvested from April to June. Methy-lation of xanthosine, 7-methylxanthine, and theobromine was catalyzed by gel-filtered leaf extracts from young shoots (April to June), but the reactions could not be detected in extracts from leaves in which no synthesis of caffeine was observed in vivo. By contrast, the activity of 5-phosphoribosyl-1-pyrophosphate synthetase was still found in leaves harvested in July and August. 

The above-mentioned facts require that ribose must have undergone further reactions immediately after its formation under prebiotic conditions. More than 20 years ago R. Shapiro (1984) pointed out the immense problems which would have needed to be solved in prebiotic nucleic acid formation. 

Although many questions are still open, peptide nucleic acids are easier to synthesize via simple reaction routes than is natural RNA. The PNAs have another important advantage they are achiral and uncharged, i.e., they contain no chiral centres in the polymeric backbone (see Sect. 9.4). Unfortunately, however, they do not fulfil all the necessary conditions for molecular information storage and transfer. Thus, the search for other possible candidates for a pre-RNA world continues. 

The importance of protonated N-hydroxy arylamines as ultimate carcinogens has been suggested for some time (28,40,139). From studies on their reactivity with nucleic acids at different pH s (2,15,16,63,130,131), the pK for protonation of the N-hydroxy group appears to be between pH 5a and 6 thus, a significant proportion (1-10%) of the N-hydroxy derivative exists as the protonated form even under neutral conditions. This would account for the significant levels of covalent modification of DNA observed in vitro by reaction with N-hydroxy arylamines at neutral pH. Consequently, it has been proposed that in vivo formation of non-acetylated aryl... 

N-AryInitrones (XIII) formed by oxidation of N-hydroxy-N-methyl arylamines, show high reactivity toward carbon-carbon and carbon-nitrogen double bonds in non-aqueous media (21,203) (Figure 10). Under physiological conditions, however, it appears that N-arylnitrones exist as protonated salts that readily hydrolyze to formaldehyde and a primary N-hydroxy arylamine and efforts to detect N-arylnitrone addition products in cellular lipid, protein or nucleic acids have not been successful (204). Nitroxide radicals derived from N-hydroxy-MAB have also been suggested as reactive intermediates (150), but their direct covalent reaction with nucleic acids has been excluded (21). 

Phenylglyoxal and alkoxyphenylglyoxals react selectively with the guanine moiety of nucleosides and nucleotides in phosphate buffer (pH 7.0) at 37°C for 5-7 min to give the corresponding fluorescent derivatives [12-15], as shown in Figure 6. Other nucleic acid bases and nucleotides (e.g., adenine, cytosine, uracil, thymine, AMP, CMP) do not produce derivatives under such mild reaction conditions. The fluorescent derivative emits chemiluminescence on oxidation with di-methylformamide (DMF) and H202 at pH 8.0-12 [14, 15],... 

The photolytic activation of 5m was also shown to lead to DNA cleavage [33,35-38]. This reaction appeared to be faster and more efficient than the Cu+-catalyzed cleavage conditions. The mechanism(s) of DNA cleavage should be different because aryl cations (not aryl radicals) are believed to be produced under photolytic conditions (Fig. 12) [7]. Such electrophiles should target the nucleic acid bases and/or the positively charged phosphodiester backbone, and both of these could lead to DNA cleavage. 

Potentially tautomeric pyrimidines and purines are /V-alkylated under two-phase conditions, using tetra-n-butylammonium bromide or Aliquat as the catalyst [75-77], Alkylation of, for example, uracil, thiamine, and cytosine yield the 1-mono-and 1,3-dialkylated derivatives [77-81]. Theobromine and other xanthines are alkylated at N1 and/or at N3, but adenine is preferentially alkylated at N9 (70-80%), with smaller amounts of the N3-alkylated derivative (20-25%), under the basic two-phase conditions [76]. These observations should be compared with the preferential alkylation at N3 under neutral conditions. The procedure is of importance in the derivatization of nucleic acids and it has been developed for the /V-alkylation of nucleosides and nucleotides using haloalkanes or trialkyl phosphates in the presence of tetra-n-butylammonium fluoride [80], Under analogous conditions, pyrimidine nucleosides are O-acylated [79]. The catalysed alkylation reactions have been extended to the glycosidation of pyrrolo[2,3-r/]pyrimidines, pyrrolo[3,2-c]pyridines, and pyrazolo[3,4-r/]pyrimidines (e.g. Scheme 5.20) [e.g. 82-88] as a route to potentially biologically active azapurine analogues. 

Before starting experiments with human or animal tissue samples, it is extremely important to optimize in vitro experimental conditions. With a purified template nucleic acid, standardize RT and PCR conditions. Check the specificity and crossreactivity of primers and probes. Sometimes it is necessary to alter MgCl2 concentration under in situ reaction conditions. The blocking reagent for filter hybridization could be different than the in situ protocol. (I use 1 % purified casein solution for filter hybridization and 3% BSA for in situ signal detection.)... 

The purines and pyrimidines are relatively stable compounds with considerable aromatic character. Nevertheless, they react with many different reagents and, under some relatively mild conditions, can be completely degraded to smaller molecules. The chemistry of these reactions is complex and is made more so by the fact that a reaction at one site on the ring may enhance the reactivity at other sites. The reactions of nucleic acids are largely the same as those of the individual nucleosides or nucleotides, the rates of reaction are often influenced by the position in the polynucleotide chain and by whether the nucleic acid is single or double stranded. The reactions of nucleosides and nucleotides are best understood in terms of the electronic properties of the various positions in the bases.26 33 Most of the chemical reactions are nucleophilic addition or displacement reactions of types that are discussed in Chapters 12 and 13. 

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