Unnatural Nucleotides Research Articles

Engineering unnatural nucleotide specificity for Rous sarcoma virus tyrosine kinase to uniquely label its direct substrates.

Protein phosphorylation plays a central role in controlling many diverse signal transduction pathways in all cells. Novel protein kinases are identified at a rapid rate using homology cloning methods and genetic screens or selections; however identification of the direct substrates of kinases has proven elusive to genetic methods because of the tremendous redundancy and overlapping of substrate specificities among protein kinases. We describe the development of a protein engineering-based method to identify the direct substrates of the prototypical protein tyrosine kinase v-Src, which controls fibroblast transformation by the Rous sarcoma virus. To differentiate the substrates of v-Src from all other kinase substrates, we mutated the ATP binding site of v-Src such that the engineered v-Src uniquely accepted an ATP analog. We show that the engineered v-Src kinase displayed catalytic efficiency with the ATP analog, N(6)-(cyclopentyl) ATP, which is similar to the wild-type kinase catalytic efficiency with ATP itself. However, the N(6)-(cyclopentyl) ATP analog was not accepted by the wild-type kinase. Furthermore, the engineered v-Src exhibited the same protein target specificity as wild-type v-Src despite the proximity of the reengineered nucleotide binding site to the phosphoacceptor binding site. The successful engineering of v-Src's active site to accept a unique nucleotide analog provides a unique handle by which the direct substrates of one kinase (v-Src) can be traced in the presence of any number of cellular kinases.

Should the asymmetry of enzymatic active centers always correlate with the asymmetry of their substrates?

Analysis of the substrate specificity of DNA polymerases and in part some other enzymes of nucleic acid metabolism towards unnatural L-stereomeric nucleosides and nucleotides was made. As a result, the hypothesis is proposed that when the chiral part of the substrate molecule does not interact specifically with the enzyme, substitution of natural D enantiomers by L compounds is possible.

Chemically modified hammerhead ribozymes with improved catalytic rates.

A site-specific chemical modification strategy has been employed to elucidate structure-function relationships at the only phylogenetically nonconserved position within the core of the hammerhead ribozyme (N7). Four different base substitutions at position 7 resulted in increased catalytic rates. A pyridin-4-one base substitution increased the rate of the chemical step up to 12-fold. These results are the first examples of chemical modifications within a catalytic RNA that enhance the rate of the chemical step. Four base substitutions resulted in decreased catalytic rates. The results do not correlate with proposed hydrogen bond interactions (Pley et al., 1994; Scott et al., 1995). This study demonstrates the utility of using unnatural nucleotide analogs-rather than mutagenesis with the four standard nucleotides alone-to elucidate structure-function relationships of small RNAs.

Unnatural Nucleotide Sequences in Biopharmaceutics

Potential therapeutic drugs and lead compounds have been identified in bacterial fermentation broths, plants, marine organisms, and cultured mammalian cells. This chapter addresses the theoretical concepts that underpin the usefulness of synthesizing vast libraries by applied molecular evolution. A comparison of the different methods will be presented; however, the focus will be on proteins with biological activity. The chapter discusses the potential use of new molecules for genetic engineering. Random sequence selection is based on the hypothesis that many amino acid sequences encode biological molecules that are more useful than those selected through evolution by nature. The idea is to insert random nucleotides in DNA and then use genetic complementation to obtain clones that encode unnatural biologically active molecules. A random sequence of nucleotides is substituted for a defined portion of a plasmid-encoded gene that specifies a biologically active molecule. In an application of this procedure, a double-stranded oligodeoxyribonucleotide is produced by hybridizing two oligonucleotides, one or both of which contain random sequences at specified positions. Prior to inserting an oligonucleotide with random sequences (vide infra) into a double-stranded DNA vector, it is necessary to excise the corresponding sequence by cutting with restriction enzymes. The most direct method for the assemblage of oligonucleotide inserts with random nucleotide sequences is to hybridize two oligonucleotides containing randomized segments and then extend them with DNA polymerase. Genetic regulatory events are governed by both protein-DNA and protein-protein interactions. Thus, in principle, random nucleotide substitutions can be carried out in either the regulatory region of genes that encode regulatory proteins. Random sequence selection, the focus of this chapter has made it feasible to select enzyme molecules with enhanced catalytic efficiency from a pool of random sequences in the absence of a detailed knowledge of the three-dimensional structure of the enzyme.

Proofreading of a mutagenic nucleotide, N 4-Aminodeoxycytidylic acid, by [formula omitted] DNA polymerase I

N 4-Aminodeoxycytidine triphosphate, a putative metabolite of N 4-aminocytidine which is a potent mutagen, is incorporated, in vitro , into polynucleotides in place of dCTP and at a much lesser extent, but significantly, in place of dTTP by E. coli DNA polymerase I large fragment. The activity of the polymerase to proofread this unnatural nucleotide has now been investigated. The results indicate that the 3′–5′ exonuclease in the polymerase recognizes N 4-aminocytosine as an incorrect base when N 4-aminocytosine is incorporated opposite adenine but the enzyme cannot distinguish N 4-aminocytosine from cytosine when it is incorporated opposite guanine.

Unnatural nucleosides and nucleotides. III. Preparation of 2‐14C and 4‐14C labelled 5‐alkyluracils and 5‐alkyl‐2′‐deoxyuridines

Abstract2‐14C Labelled 5‐alkyluracils were prepared by condensation of the diethylacetals of α‐formyl‐carbonic acid esters with 14C‐thiourea. Compounds labelled at 4‐C were synthesized by condensation of the labelled carboxylic acid derivatives with thiourea.β‐Anomers of 5‐alkyl‐2′‐deoxyuridines were obtained in a fairly good radiochemical yield. Alkyl substituents ranged from methyl to tetradecyl, isopropyl and tertbutyl.

Polyphosphate Nucleosides of Purine Analogues

(Received for publication, November 17, 1958) To test the hypothesis that carcinostatic purine and pyrimi- dine analogues exert their effects at the nucleotide level, we have undertaken the preparation and study of nucleotides, including coenzymes, which contain analogues in place of the natural bases. An earlier communication from this laboratory (1) de- scribed the enzymatic synthesis and isolation of 5’-nucleotides of a number of purine analogues. The present report is con- cerned with the preparation of the polyphosphate nucleosides. This has been accomplished chemically by use of the carbodi- imide procedure (2) and enzymatically with enzymes from beef liver and pork kidney. Most of the studies described below employ the unnatural nucleotide, 8azaguanosine 5’-phosphate.