Nucleic Acid-catalyzed Reactions

Nucleic acids catalyze many different types of reactions. Some RNA-catalyzed transformations show stereoselectivity [10,34]. The potential scope of organic reactions is quite broad, with a commensurate variability in reaction conditions. The essential components present in successful nucleic acid-catalyzed reactions are divalent metal ions such as Mg2+, Ca2+, Cu2+, Zn2+, as well as K+ [7,10,21,35,36]. A buffer is also required but should not contain functional groups that are reactive under the reaction conditions. A commonly used buffer is HEPES (2-[4-(2-hydroxyethyl)-l-piperazine]ethanesulfonic acid). These essential components are present to maintain the RNA s tertiary structure and prevent its aggregation. Because these reactions are carried out in aqueous solution, the addition of a co-solvent (e. g., DM SO or EtOH) may be necessary, depending on the solubility of the substrates. 

Add BMCC-biotin to a final concentration of 100 pM (its addition accounts for the total amount of 2% DMSO in the reaction mix) 

Dilute the reaction mixture by adding 4 volumes of H20. Remove unreacted BMCC-biotin through a 30kDaMWCO filter (Millipore) by centrifugation for 8 min at 11 000 x g. Repeat this step to ensure that all unreacted BMCC has been removed before proceeding to the partitioning step (Section 8.3.6). 

---

A new departure in the development of nucleic acid based catalysts was made three years ago by Breaker and Joyce, who reported the isolation of the first deoxyribozyme. The proof that DNA can also catalyze chemical reactions is not completely unexpected, especially since it was shown shortly after the development of the in vitro selection technique that single-stranded DNA molecules can also be selected to bind to a variety of ligands. Meanwhile, several catalytically active DNAs have been described, expanding the range of nucleic acid catalyzed reactions even further. 

Plants and animals synthesize a number of polymers (e.g., polysaccharides, proteins, nucleic acids) by reactions that almost always require a catalyst. The catalysts present in living systems are usually proteins and are called enzymes. Reactions catalyzed by enzymes are called enzymatic reactions, polymerizations catalyzed by enzymes are enzymatic polymerizations. Humans benefit from naturally occurring polymers in many ways. Our plant and animal foodstuffs consist of these polymers as well as nonpolymeric materials (e.g., sugar, vitamins, minerals). We use the polysaccharide cellulose (wood) to build homes and other structures and to produce paper. 

Water is not just the solvent in which the chemical reactions of living cells occur it is very often a direct participant in those reactions. The formation of ATP from ADP and inorganic phosphate is an example of a condensation reaction in which the elements of water are eliminated (Fig. 2-22a). The reverse of this reaction— cleavage accompanied by the addition of the elements of water—is a hydrolysis reaction. Hydrolysis reactions are also responsible for the enzymatic depolymerization of proteins, carbohydrates, and nucleic acids. Hydrolysis reactions, catalyzed by enzymes called... 

RNA replicase isolated from Qj8-infected E. coli cells catalyzes the formation of an RNA complementary to the viral RNA, in a reaction equivalent to that catalyzed by DNA-dependent RNA polymerases. New RNA strand synthesis proceeds in the 5 —>3 direction by a chemical mechanism identical to that used in all other nucleic acid synthetic reactions that require a template. RNA replicase requires RNA as its template and will not function with DNA. It lacks a separate proofreading endonuclease activity and has an error rate similar to that of RNA polymerase. Unlike the DNA and RNA polymerases, RNA replicases are specific for the RNA of their own virus the RNAs of the host cell are generally not replicated. This explains how RNA viruses are preferentially replicated in the host cell, which contains many other types of RNA. 

Aspartate transcarbamoylase catalyzes the first step in the biosynthesis of pyrimidines, bases that are components of nucleic acids. The reaction catalyzed by this enzyme is the condensation of aspartate and carbamoyl phosphate to form A-carbamoylaspartate and orthophosphate (Figure 10.1). ATCase catalyzes the committed step in the pathway that will ultimately yield pyrimidine nucleotides such as cytidine triphosphate (CTP). How is this enzyme regulated to generate precisely the amount of CTP needed by the cell ... 

Quinone methides have been shown to be important intermediates in chemical synthesis,1 2 in lignin biosynthesis,3 and in the activity of antitumor and antibiotic agents.4 They react with many biologically relevant nucleophiles including alcohols,1 thiols,5-7 nucleic acids,8-10 proteins,6 11 and phosphodiesters.12 The reaction of nucleophiles with ortho- and /iara-quinone methides is pH dependent and can occur via either acid-catalyzed or uncatalyzed pathways.13-17 The electron transfer chemistry that is typical of the related quinones does not appear to play a role in the nucleophilic reactivity of QMs.18... 

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 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. 

Virtually all biological reactions are stereospecific. This generalization applies not only to the enzyme-catalyzed reactions of intermediary metabolism, but also to the processes of nucleic acid synthesis and to the process of translation, in which the amino acids are linked in specific sequence to form the peptide chains of the enzymes. This review will be restricted mainly to some of the more elementary aspects of the stereospecificity of enzyme reactions, particularly to those features of chirality which have been worked out with the help of isotopes. 

While indirect selections work quite well for antibodies they have been less successful in the case of catalytic nucleic acids. There are only three examples which prove that it is possible in principle to obtain a ribo- or deoxyribozyme by selecting an aptamer that binds to a TSA A rotamase ribozyme [7], a ribozyme capable of catalyzing the metallation of a porphyrin derivative [92], and one catalytic DNA of the same function [93]. Another study reported the selection of a population of RNA-aptamers which bind to a TSA for a Diels-Alder reaction but the subsequent screen for catalytic activity was negative for all individual RNAs tested [94]. The attempt to isolate a transesterase ribozyme using the indirect approach also failed [95]. 

The more successful strategy for the isolation of RNA- and DNA-based catalysts involves the direct screening of nucleic acids libraries for catalytic activity. This approach is called direct selection [6, 65, 77, 78, 86, 101-107]. In direct selections, nucleic acids that are capable of catalyzing a particular chemical transformation modify themselves with a tag or other characteristic that allows their preferential enrichment over those molecules which are catalytically inactive [108]. The design of ribozyme-selections involving reactions between two small substrates requires that one reactant be covalently attached to every individual member of the starting RNA pool. After the reaction with another substrate which usually carries the selection-tag has occurred, the self-modified RNA is immobilized on a solid support, separated from non-active molecules, and then cleaved off the support. 

Many examples of catalytic nucleic acids obtained by in vitro selection demonstrate that reactions catalyzed by ribozymes are not restricted to phosphodiester chemistry. Some of these ribozymes have activities that are highly relevant for theories of the origin of life. Hager et al. have outlined five roles for RNA to be verified experimentally to show that this transition could have occurred during evolution [127]. Four of these RNA functionalities have already been proven Its ability to specifically complex amino acids [128-132], its ability to catalyze RNA aminoacylation [106, 123, 133], acyl-transfer reactions [76, 86], amide-bond formation [76,77], and peptidyl transfer [65,66]. The remaining reaction, amino acid activation has not been demonstrated so far. 

A new dimension in the development of nucleic acid based catalysts was introduced by Breaker and Joyce in 1994 when they isolated the first deoxyribozyme [111]. It is not unexpected that DNA is also able to catalyze chemical reactions because it was shown previously that ssDNA aptamers which bind to a variety of ligands can be isolated by in vitro selection [141]. In the meantime, several deoxyribozymes have been described which expand the range of chemical transformations accelerated by nucleic acid catalysts even further and raising question whether even catalytic DNA might have played some role in the pre-biotic evolution of hfe on earth [69-71]. 

Stereochemical probes of the specificity of substrates, products, and effectors in enzyme-catalyzed reactions, receptor-ligand interactions, nucleic acid-ligand interactions, etc. Most chirality probe studies attempt to address the stereospecificity of the substrates or ligands or even allosteric effectors. However, upon use of specific kinetic probes, isotopic labeling of achiral centers, chronfium-or cobalt-nucleotide complexes, etc., other stereospecific characteristics can be identified, aU of which will assist in the delineation of the kinetic mechanism as well as the active-site topology. A few examples of chirality probes include ... 

Although some depolymerases act processively in cleaving their polymeric substrates, others act by what can be described as multiple attack which results in nonselective scission or random scission. The analysis of cleavage products during the course of enzyme-catalyzed depolymerization can provide important clues about the nature of the reaction. With random scission, the rate of bond scission must be proportional to the total number of unbroken bonds present in the solution. Thomas measured the rate of base addition in a pH-Stat (a device with an automated feedback servomotor that expels ti-trant from a syringe to maintain pH) to follow the kinetics of DNA bond scission by DNase. The number of bonds cleaved was linear with time, and this was indicative of random scission. In other cases, one may apply the template challenge method to assess the processivity of nucleic acid polymerases. See Processivity... 

Metal ions, especially Zn(II), play an important role in many enzyme-catalyzed reactions involving nucleic acids, such as DNA cleavage by zinc nuclease. Therefore, the binding of Zn(II) to a 19-mer double-stranded oligodeoxyribonucleotide was investigated to understand the role of zinc in DNA cleavage catalyzed by mung bean nuclease [107]. 

---