A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the -17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3' terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.

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A 19-nucleotide RNA fragment can cause rapid, highly specific cleavage of a 24-nucleotide RNA fragment under physiological conditions. Because each 19-mer can participate in many cleavage reactions, this molecule has all the properties associated with an RNA enzyme.

A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study. The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined. Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage. In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex. In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing an alternate substrate or ribozyme secondary structure. All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA. The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences.

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A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNAPhe 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 tRNAPhe transcript can be specifically aminoacylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNAPle.The Km is independent of Mg2+ concentration, whereas the V,.. is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNAPhe at different Mg2+ concentrations suggests that the unmodified tRNAPhe 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 Vma./Km by a factor of 14 for aminoacylation of the mutant tRNAP`" transcript is observed.A useful approach for understanding the relationship between structure and function of tRNA involves the physical and biochemical analysis of variant tRNA molecules. A number of methods have been used to create mutant tRNAs in which one or more nucleotides have been altered. Various mutagenesis techniques have produced a large number of mutant tRNA genes (1, 2), but often the expression of the mutant gene is blocked at transcription or processing steps such that biochemical amounts of the tRNA cannot be isolated. tRNAs having specific nucleotide substitutions in the anticodon loop and the TTCG loop have been prepared by the removal of these nucleotides with RNase followed by the insertion of an altered oligoribonucleotide with RNA ligase (3,4). While this approach has produced many valuable data, specific substitution is limited to those regions of the molecule that are susceptible to partial nuclease digestion. Complete synthesis of two tRNAs has been achieved by joining synthetic oligoribonucleotides with RNA ligase (5,6). Although this would in principle allow for the synthesis of any desired tRNA sequence, the multiple ligations and subsequent purification of intermediates result in a low yield of final product.It has been shown that the 3' terminus of brome mosaic virus RNA3, synthesized by in vitro runoff transcription of cloned DNA, can be specifically aminoacylated by tyrosyltRNA synthetase (7). It therefore seemed clear that a tRNA lacking the modified nucleotides could be synthesized in a similar manner. In this paper, we describe a detailed method for the synthesis of an unmodified yeast tRNAPhC by runoff transcription using T7 RNA polymerase. The aminoacylation properties of the wild-type tRNAPhC transcript and a mutant transcript will be discussed. A preliminary account of some of this work has been reported earlier (8). (p67YFO) were sequenced in the region of the insert with reverse transcriptase using either the oligomer corresponding to the top strand of the T7 pro...

SELEX is a technology for the identification of high affinity oligonucleotide ligands. Large libraries of random sequence single-stranded oligonucleotides, whether RNA or DNA, can be thought of conformationally not as short strings but rather as sequence dependent folded structures with high degrees of molecular rigidity in solution. This conformational complexity means that such a library is a source of high affinity ligands for a surprising variety of molecular targets, including nucleic acid binding proteins such as polymerases and transcription factors, non-nucleic acid binding proteins such as cytokins and growth factors, as well as small organic molecules such as ATP and theophylline. The range of applications of this technology for new discovery extends from basic research reagents to the identification of novel diagnostic and therapeutic reagents. Examples of these applications are described along with a discussion of underlying principles and future developments expected to further the utility of SELEX.