High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.

https://science.sciencemag.org/content/sci/249/4968/505.full.pdf

Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase

We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 μg/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor transfer RNA (tRNA).

/pdf/cell-free-translation-reconstituted-with-purified-components-3yqtgmhg62.pdf

Cell-free translation reconstituted with purified components

Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA

A new method has been developed that makes it possible to site-specifically incorporate unnatural amino acids into proteins. Synthetic amino acids were incorporated into the enzyme beta-lactamase by the use of a chemically acylated suppressor transfer RNA that inserted the amino acid in response to a stop codon substituted for the codon encoding residue of interest. Peptide mapping localized the inserted amino acid to a single peptide, and enough enzyme could be generated for purification to homogeneity. The catalytic properties of several mutants at the conserved Phe66 were characterized. The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.

https://science.sciencemag.org/content/sci/244/4901/182.full.pdf

A general method for site-specific incorporation of unnatural amino acids into proteins.

RNA enzymes or ribozymes can act as endoribonucleases, catalyzing the cleavage of RNA molecules with a sequence specificity of cleavage greater than that of known ribonucleases and approaching that of the DNA restriction endonucleases, thus serving as RNA sequence specific endoribonucleases. An example is a shortened form of the self-splicing ribosomal RNA intervening sequence of Tetrahymena (L-19 IVS RNA). Site-specific mutagenesis of the enzyme active site of the L-19 IVS RNA alters the substrate sequence specificity in a predictable manner, allowing a set of sequence-specific endoribonucleases to be synthesized. Varying conditions allow the ribozyme to act as a polymerase (nucleotidyltransferase), a dephosphorylase (acid phosphatase or phosphotransferase) or a sequence-specific endoribonuclease.

Rna ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods.

A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNA(Phe) is flanked by a T7 promoter and a BstNI restriction site. Runoff transcription of the BstNI-digested plasmid with T7 RNA polymerase gives an unmodified tRNA of the expected sequence having correct 5' and 3' termini. This tRNA(Phe) transcript can be specifically aminoacylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNA(Phe). The Km is independent of Mg2+ concentration, whereas the Vmax is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNA(Phe) at different Mg2+ concentrations suggests that the unmodified tRNA(Phe) has a less stable tertiary structure. Using one additional DNA oligomer, a mutant plasmid was constructed having a guanosine to thymidine change at position 20 in the tRNA gene. A decrease in Vmax/Km by a factor of 14 for aminoacylation of the mutant tRNA(Phe) transcript is observed.

https://www.pnas.org/content/pnas/85/4/1033.full.pdf

Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro.

An analysis of the aminoacylation kinetics of unmodified yeast tRNAPhe mutants revealed that five single-stranded nucleotides are important for its recognition by yeast phenylalanyl-tRNA synthetase, provided they were positioned correctly in a properly folded tRNA structure. When four other tRNAs were changed to have these five nucleotides, they became near-normal substrates for the enzyme.

http://science.sciencemag.org/content/sci/243/4896/1363.full.pdf

Nucleotides in yeast tRNAPhe required for the specific recognition by its cognate synthetase.

Yeast tRNA(Phe) lacking modified nucleotides undergoes lead-catalyzed cleavage between nucleotides U17 and G18 at a rate very similar to that of its fully modified counterpart. The rates of cleavage for 28 tRNA(Phe) mutants were determined to define the structural requirements of this reaction. The cleavage rate was found to be very dependent on the identity and correct positioning of the two lead-coordinating pyrimidines defined by X-ray crystallography. Nucleotide changes that disrupted the tertiary interactions of tRNAPhe reduced the rate of cleavage even when they were distant from the lead binding pocket. However, nucleotide changes designed to maintain tertiary interactions showed normal rates of cleavage, thereby making the reaction of a useful probe for tRNA(Phe) structure. Certain mutants resulted in the enhancement of cleavage at a "cryptic" site at C48. The sequences of Escherichia coli tRNA(Phe) and yeast tRNA(Arg) were altered such that they acquired the ability to cleave at U17, confirming our understanding of the structural requirements for cleavage. This mutagenic analysis of the lead cleavage domain provides a useful guide for similar analysis of autocatalytic self-cleavage reactions.

Lead-catalyzed cleavage of yeast tRNAPhe mutants.

The authors have used NMR to study the structure of the yeast tRNA{sup Phe} sequence which was synthesized by using T7 RNA polymerase. Many resonances in the imino {sup 1}H spectrum of the transcript have been assigned, including those of several tertiary interactions. When the Mg{sup 2+} concentration is high, the transcript appears to fold normally, and the spectral features of the transcript resemble those of tRNA{sup Phe}. The transcript has been shown to be aminoacylated with kinetics similar to the modified tRNA{sup Phe} suggesting that the structure of the two molecules must be similar. In the absence of Mg{sup 2+} or at (tRNA):(Mg{sup 2+}) ratios <0.2, the transcript does not adopt the native structure, as shown by both chemical shifts and NOE patterns. In these low Mg{sup 2+} conditions, a second GU base pair is found, suggesting a structural rearrangement of the transcript. NMR data indicate that the structure of a mutant having G20 changed to U20 is nearly identical with that of the normal sequence, suggesting that the low aminoacylation activity of this variant is not due to a substantially different conformation.

Structure of an unmodified tRNA molecule.

In vitro transcription by T7 RNA polymerase was used to prepare 32 different mutations in the 21 nucleotides that participate in the 9 tertiary base pairs or triples of yeast tRNAPhe. The mutations were designed either to disrupt the tertiary interaction or to change the sequence without disrupting the structure by transplanting tertiary interactions present in other tRNAs. Steady-state aminoacylation kinetics with purified yeast phenylalanyl synthetase revealed little change in reaction rate as long as a tertiary interaction was maintained. This suggests that the tertiary nucleotides only contribute to the folding of tRNAPhe and do not participate directly in sequence-specific interaction with the synthetase.

Jeffrey R. Sampson

Papers

Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro.

Nucleotides in yeast tRNAPhe required for the specific recognition by its cognate synthetase.

Lead-catalyzed cleavage of yeast tRNAPhe mutants.

Structure of an unmodified tRNA molecule.

Role of the tertiary nucleotides in the interaction of yeast phenylalanine tRNA with its cognate synthetase.