Potent and selective gene inhibition using antisense oligodeoxynucleotides

The development of antisense technology as a generally useful tool relies on the use of potent agents and the utilization of many controls in experiments. Here we describe our experience using oligodeoxynucleotides (ODNs) containing C-5 propynyl pyrimidine and phosphorothioate modifications as broadly applicable gene inhibition agents in cell culture. Methods include selection of antisense sequences, synthesis and purification of ODNs, choice of controls, delivery methods (microinjection, cationic lipid transfection, and electroporation), and analysis of gene inhibition.     

Inducing nonsense suppression by targeted pseudouridylation

Isomerization from uridine to pseudouridine (pseudouridylation) is largely catalyzed by a family of small ribonucleoproteins called box H/ACA RNPs, each of which contains one unique small RNA-the box H/ACA RNA. The specificity of the pseudouridylation reaction is determined by the base-pairing interactions between the guide sequence of the box H/ACA RNA and the target sequence within an RNA substrate. Thus, by creating a new box H/ACA RNA harboring an artificial guide sequence that base-pairs with the substrate sequence, one can site-specifically introduce pseudouridines into virtually any RNA (e.g., mRNA, ribosomal RNA, small nuclear RNA, telomerase RNA and so on). Pseudouridylation changes the properties of a uridine residue and is likely to alter the role of its corresponding RNA in certain cellular processes, thereby enabling basic research into the effects of RNA modifications. Here we take a TRM4 reporter gene (also known as NCL1) as an example, and we present a protocol for designing a box H/ACA RNA to site-specifically pseudouridylate TRM4 mRNA. Disease-related mutation can result in early termination of translation by creating a premature termination codon (PTC); however, pseudouridylation at the PTC can suppress this translation termination (nonsense suppression). Thus, the experimental procedures described in this protocol may provide a novel way to treat PTC-related diseases. This protocol takes 10-13 d to complete.     

Genetic studies on temperature sensitive nonsense suppression

Summary Temperature sensitive suppression of nonsense mutations has been obtained, in E. coli, by inducing a mutation in the suppressor gene su-4, a structural gene for a tyrosine tRNA. In this mutant while no suppression is detectable at 42°C it is always present at lower temperatures. This temperature sensitivity is amenable to a reversible inactivation of the product of the su-4 gene.     

Gene interference using antisense oligodeoxynucleotides on whole chick embryos

Details are given of an advanced version of the ring method of chick embryo culture. This ensures good development from early blastoderm stages even when the culturing procedure is interrupted by the extended periods required for collecting matched embryo samples and for preparing antisense treatment. Antisense oligodeoxynucleotide treatment, in short-term incubation before return of blastoderms to their ring cultures, is then described. An alternative, roller-bottle, culture method for continued development after treatment is also described. Criteria for the validity and success of this gene interference method are given. While the toxt is meant to be of detailed prootioal help to these inexperienced in ombryo culture a preliminary reading, and familiarity with its sectional (Subheading) structure, is recommended before work is undertaken.     

Gene transfer in staphylococci: stimulation and suppression by antibacterial agents

The Editors of Letters in Applied Microbiology will, at their discretion, publish invited and submitted 'Opinions' on subjects in the general area of Applied Microbiology. They will not be subjected to the normal refereeing procedures and reprints will not be provided. The 'Opinions' will not necessarily represent the views of the Society for Applied Bacteriology or of the Editors. The Editors may invite or readers may submit 'Responses' to published 'Opinions' , provided that they are not merely polemics, and these will also be published at the discretion of the Editors. 'Responses' will be treated precisely like 'Opinions' . They should clearly indicate, in their first sentence, the 'Opinion' to which they are a response.     

Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs

Translational stop codon readthrough provides a regulatory mechanism of gene expression that is extensively utilised by positive-sense ssRNA viruses. The misreading of termination codons is achieved by a variety of naturally occurring suppressor tRNAs whose structure and function is the subject of this survey. All of the nonsense suppressors characterised to date (with the exception of selenocysteine tRNA) are normal cellular tRNAs that are primarily needed for reading their cognate sense codons. As a consequence, recognition of stop codons by natural suppressor tRNAs necessitates unconventional base pairings in anticodon–codon interactions. A number of intrinsic features of the suppressor tRNA contributes to the ability to read non-cognate codons. Apart from anticodon–codon affinity, the extent of base modifications within or 3′ of the anticodon may up- or down-regulate the efficiency of suppression. In order to out-compete the polypeptide chain release factor an absolute prerequisite for the action of natural suppressor tRNAs is a suitable nucleotide context, preferentially at the 3′ side of the suppressed stop codon. Three major types of viral readthrough sites, based on similar sequences neighbouring the leaky stop codon, can be defined. It is discussed that not only RNA viruses, but also the eukaryotic host organism might gain some profit from cellular suppressor tRNAs.     

Effects of surrounding sequence on the suppression of nonsense codons

Using a lacI-Z fusion system, we have determined the efficiency of suppression of nonsense codons in the I gene of Escherichia coli by assaying beta-galactosidase activity. We examined the efficiency of four amber suppressors acting on 42 different amber (UAG) codons at known positions in the I gene, and the efficiency of a UAG suppressor at 14 different UGA codons. The largest effects were found with the amber suppressor supE (Su2), which displayed efficiencies that varied over a 35-fold range, and with the UGA suppressor, which displayed a 170-fold variation in efficiency. Certain UGA sites were so poorly suppressed (less than 0.2%) by the UGA suppressor that they were not originally detected as nonsense mutations. Suppression efficiency can be correlated with the sequence on the 3' side of the codon being suppressed, and in many cases with the first base on the 3' side. In general, codons followed by A or G are well suppressed, and codons followed by U or C are poorly suppressed. There are exceptions, however, since codons followed by CUG or CUC are well suppressed. Models explaining the effect of the surrounding sequence on suppression efficiency are considered in the Discussion and in the accompanying paper.     

Facile characterization of translation initiation via nonsense codon suppression.

A new strategy for studying the mechanism of translation initiation in eukaryotes has been developed. The strategy involves the use of an in vitro translation system to incorporate a non-natural fluorescent amino acid into a protein from a suppressor tRNAPheCUA misacylated with that amino acid. It is thereby possible to monitor translation initiation efficiency at an AUG codon in different contexts; this is illustrated for three constructs encoding Escherichia coli dihydrofolate reductase mRNA with different translation initiation regions. Fluorescence measurements after in vitro translation of the mRNAs in rabbit reticulocyte lysate reflected differences in the position and efficiency of translation initiation and, therefore, can be used for characterization of the translation initiation process.     

Allele-specific therapy: suppression of nonsense mutations by readthrough inducers

Ten percent of human hereditary diseases are linked to nonsense mutations (premature termination codon). These mutations lead to premature translation termination, trigger the synthesis of a truncated protein and possibly lead to mRNA degradation by the NMD pathway (nonsense mediated mRNA decay). For the past ten years, therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at premature stop codon (readthrough), thus allowing for the synthesis of a full length protein. Molecules currently used for this approach are mostly aminoglycoside antibiotics (gentamicin, amikacin…) that bind the decoding center of the ribosome. This therapeutic approach has been studied for various genetic diseases including Duchenne muscular dystrophy (DMD) and cystic fibrosis. The feasibility of this approach depends on induced readthrough level, mRNA quantity, re-expressed protein functionality and characteristics of each disease.     

Analysis of btuB receptor function by use of nonsense suppression.

Informational suppression of btuB nonsense mutants allows the study of the effect of known, single amino acid substitutions on receptor function. We found that ligand uptake is largely unaffected by such amino acid changes. The few instances in which certain substitutions destroyed sensitivity to the two lethal agents (phage BF23, colicin E3) without affecting vitamin B12 uptake suggest a common region on the btuB receptor involved in the binding of these proteinaceous agents.     

The requirement of nonsense suppression for the development of several phages

Summary A spontaneous streptomycin-resistant Escherichia coli mutant which is temperature-sensitive for suppression of a nonsense codon was studied for its ability to propagate phages T2, T4D, T5, K, f2, MS2, R17, Q, as well as filamentous phages fl, fd and M13. Of all phages tested, only the growth of Q, , and filamentous phages is inhibited in the mutant at 42° C. This selective inhibition suggests that, like Q, and filamentous phages also require a read-through protein(s) which results from suppression of a termination codon.     

Toxicity of diphtheria toxin-related proteins produced by suppression of nonsense mutations

The Mr = 62,000 diphtheria toxin-related proteins produced from the suppression of nonsense mutations within the tox gene of corynephage beta were purified by affinity chromatography. Except for the toxin 111-sup2-62, the Mr = 62,000 polypeptides were found to have the same specific toxicity as does wild type toxin. 111-sup2-62 was found to have a prolonged lag period prior to the onset of inhibition of protein synthesis and ADP-ribosylation of elongation factor 2. 111-sup2-62 differs from wild type toxin by an amino acid substitution at a site approximatley 47,000 daltons from the NH2 terminus. The data presented provide genetic support for the Boquet-Pappenheimer model (Boquet, P., and Pappenheimer, A. M. Jr (1978) J. Biol. Chem. 251, 5770-5778) of fragment A translocation into the eukaryotic cell cytosol.     

In vitro suppression of a nonsense mutant of Drosophila melanogaster.

When RNA isolated from the Drosophila melanogaster alcohol dehydrogenase (ADH) negative mutant CyOnB was translated "in vitro" in the presence of yeast opal suppressor tRNA, a wild type size ADH protein was obtained in addition to the mutant gene product. This identifies the CyOnB mutant as an opal (UGA) nonsense mutant. From the molecular weight of the mutant protein, and from the known sequence of the ADH gene (Benyajati et al., Proc.Natl.Acad.Sci. USA 78, 2717-2721, 1981), we conclude that the tryptophan codon UGG in position 234 has been changed into a UGA nonsense codon in the CyOnB mutant. Furthermore, we show that the UAA stop codon of the wild type ADH gene is resistant to suppression by a yeast ochre suppressor tRNA. This is in contrast to the high efficiency of suppression of the CyOnB UGA nonsense codon, despite an almost identical codon context.     

Purification of plasmid DNA using selective precipitation by compaction agents.

A scaleable method for the liquid-phase separation of plasmid DNA from RNA.     

The immunological detection of yeast nonsense termination fragments on sodium dodecylsulfate-polyacrylamide gels.

A sensitive immunological technique is described that detects proteins in sodium dodecylsulfate gels of crude cell extracts. The method is based on the binding of ¹²⁵I-protein A to gels that have been incubated with antibody to a specific protein. Using antibody to the yeast $\text{HIS4}$ protein, single polypeptides can be detected in mutant and wild-type extracts. The size of these polypeptides correlates both with the type of mutation and with its location in the $\text{HIS4,m}$ region.     

Mechanism for suppression mRNA translation with antisense oligonucleotides

Experimental studies of the effects of antisense oligonucleotides on translation of mRNAs in cell-free systems are reviewed. Oligonucleotides complementary to the leader sequences or to the sequence overlapping the initiating codon region of mRNAs inhibit translation of the messengers. In the presence of ribonuclease H, oligodeoxyribonucleotides and their phosphorothioate analogs complementary either to the mentioned mRNA regions or to the mRNA coding sequence suppress the translation due to the RNAs cleavage. This inhibition-enhancing mechanism does not operate in the case of the oligonucleotide analogs--oligonucleoside methylphosphonates and oligonucleotides built of the alpha-nucleosides, since the complexes formed by RNA and these analogs are not substrates of the ribonuclease H. The translation inhibition efficiency is determined by the oligonucleotides lengths and by the availability of the complementary sequence in the mRNA structure. The oligonucleotides inhibitory power can be improved by the coupling to the oligonucleotides of the intercalating groups and the reactive groups.     

NPY-induced feeding: pharmacological characterization using selective opioid antagonists and antisense probes in rats

The ability of neuropeptide Y to potently stimulate food intake is dependent in part upon the functioning of mu and kappa opioid receptors. The combined use of selective opioid antagonists directed against mu, delta or kappa receptors and antisense probes directed against specific exons of the MOR-1, DOR-1, KOR-1 and KOR-3/ORL-1 opioid receptor genes has been successful in characterizing the precise receptor subpopulations mediating feeding elicited by opioid peptides and agonists as well as homeostatic challenges. The present study examined the dose-dependent (5-80 nmol) cerebroventricular actions of general and selective mu, delta, and kappa1 opioid receptor antagonists together with antisense probes directed against each of the four exons of the MOR-1 opioid receptor gene and each of the three exons of the DOR-1, KOR-1, and KOR-3/ORL-1 opioid receptor genes upon feeding elicited by cerebroventricular NPY (0.47 nmol, 2 ug). NPY-induced feeding was dose-dependently decreased and sometimes eliminated following pretreatment with general, mu, delta, and kappa1 opioid receptor antagonists. Moreover, NPY-induced feeding was significantly and markedly reduced by antisense probes directed against exons 1, 2, and 3 of the MOR-1 gene, exons 1 and 2 of the DOR-1 gene, exons 1, 2, and 3 of the KOR-1 gene, and exon 3 of the KOR-3/ORL-1 gene. Thus, whereas the opioid peptides, beta-endorphin and dynorphin A(1-17) elicit feeding responses that are respectively more dependent upon mu and kappa opioid receptors and their genes, the opioid mediation of NPY-induced feeding appears to involve all three major opioid receptor subtypes in a manner similar to that observed for feeding responses following glucoprivation or lipoprivation.