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...

CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence (‘guide sequence’) and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2′-O-methyl-3′-phosphonoacetate, or ‘MP’) incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.

We present here the first fully integrated, comprehensive map of the canine genome, incorporating detailed cytogenetic, radiation hybrid (RH), and meiotic information. We have mapped a collection of 266 chromosome-specific cosmid clones, each containing a microsatellite marker, to all 38 canine autosomes by fluorescence in situ hybridization (FISH). A 1500-marker RH map, comprising 1078 microsatellites, 320 dog gene markers, and 102 chromosome-specific markers, has been constructed using the RHDF5000-2 whole-genome radiation hybrid panel. Meiotic linkage analysis was performed, with at least one microsatellite marker from each dog autosome on a panel of reference families, allowing one meiotic linkage group to be anchored to all 38 dog autosomes. We present a karyotype in which each chromosome is identified by one meiotic linkage group and one or more RH groups. This updated integrated map, containing a total of 1800 markers, covers >90% of the dog genome. Positional selection of anchor clones enabled us, for the first time, to orientate nearly all of the integrated groups on each chromosome and to evaluate the extent of individual chromosome coverage in the integrated genome map. Finally, the inclusion of 320 dog genes into this integrated map enhances existing comparative mapping data between human and dog, and the 1000 mapped microsatellite markers constitute an invaluable tool with which to perform genome scanning studies on pedigrees of interest.

The nonsense codon suppression method for unnatural amino acid incorporation has been applied to intact cells and combined with electrophysiological analysis to probe structure-function relations in the nicotinic acetylcholine receptor. Functional receptors were expressed in Xenopus oocytes when tyrosine and phenylalanine derivatives were incorporated at positions 93, 190, and 198 in the binding site of the alpha subunit. Subtle changes in the structure of an individual side chain produced readily detectable changes in the function of this large channel protein. At each position, distinct features of side chain structure dominated the dose-response relation, probably by governing the agonist-receptor binding.

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.