RDL A301S alone does not confer high levels of resistance to cyclodiene organochlorine or phenyl pyrazole insecticides in Plutella xylostella.

Mutations in the GABA-gated chloride channel are associated with resistance to cyclodiene organochlorine and phenyl pyrazole insecticides. The best characterised of these is A301S, which was initially identified in a Dieldrin resistant strain of Drosophila melanogaster. The orthologous mutation has been found in a variety of different crop pests including the diamond back moth Plutella xylostella. However, the contribution of this mutation to resistance in this species remains unclear. We have used the CRISPR/Cas9 system in order to edit Plutella xylostella PxGABARalpha1 to Serine at the 301 orthologous position (282 in PxGABARalpha1) in an insecticide sensitive strain isolated from Vero Beach (VB) USA. In this edited line, no high level of resistance is conferred to Dieldrin, Endosulfan or Fipronil, rather only a subtle shift in sensitivity which could not confer commercially important resistance. We conclude that the high level of commercial resistance to cyclodiene organochlorine and phenyl pyrazole insecticides observed in some field isolates of Plutella xylostella cannot arise from A282S in PxGABARalpha1 alone.

Dens evaginatus and dens invaginatus in a maxillary lateral incisor: report of a rare occurrence and review of literature.

A case of dens evaginatus (DE) and dens invaginatus (DI) concurrently affecting the maxillary right permanent lateral incisor in a 25-year-old Hispanic male is reported. DE, referred to as Talon's cusp in the anterior teeth and Leong's premolar in the premolar teeth, is a relatively rare condition by itself. An association of DI with this rare anomaly within the same tooth has never been reported before although it has been known to occur within the same patient. Since it is known that DE may be composed of normal enamel and dentine, as well as varying amounts of pulpal tissue, care should be exercised while performing any aesthetic procedures to remove or recontour it.

Optimization of hammerhead ribozymes for the cleavage of S100A4 (CAPL) mRNA.

Previously, suppression of the S100A4 mRNA by an endogenously expressed ribozyme in osteosarcoma cells was shown to inhibit their metastasis in rats. As a prelude to performing similar studies with exogenous, synthetic ribozymes, we compared a series of hammerhead ribozymes targeted against different sites in the mRNA. The ribozymes differed only in the 7-base flanking sequences complementary to the substrate and were protected against nucleases by chemical modification. Cleavage efficiency varied widely and was not obviously related to the predicted secondary structure of the target RNA. The most active ribozyme of the series was chosen for further optimization. Lengthening its flanking sequences was counterproductive and reduced cleavage even when using excess ribozyme. Using excess substrate (multiple-turnover kinetics), cleavage was fastest with the (6+8) ribozyme having 6 nucleotides (nt) in stem III and 8 nt in stem I. Although these stems strongly influence ribozyme performance, their optimization is still empirical. Faster cleavage was obtained by adding facilitator oligonucleotides to ribozymes with shorter stems of (6+6) and (5+5) nt. Stimulation was particularly strong in the case of the (5+5) ribozyme, which was poorly active by itself. The enhancement caused by different facilitator oligonucleotides paralleled their expected ability to hybridize to RNA as a function of length and chemical modification.

Hammerhead ribozymes: biochemical and chemical considerations.

Ribozymes can be used to cleave specific mRNAs so as to prevent their translation. This presents an alternative to the use of antisense oligonucleotides for drug target validation or therapeutic purposes. The present review covers only one class of ribozymes, the hammerheads, which are small enough for chemical synthesis. Following an account of their structure and ability to catalyze the cleavage of target RNA is a discussion of some problems associated with their use as exogenous therapeutics. Chemical modifications that are used to address these issues are described.

Cancer immunotherapy by antisense suppression of Ii protein in MHC-class-II-positive tumor cells.

This study was aimed at creating a more effective tumor cell vaccine by suppressing Ii protein in the presence of MHC class II molecules within a cancer cell. Absence of the Ii protein, which normally blocks the antigenic-peptide-binding site of MHC class II molecules at synthesis in the endoplasmic reticulum, presumably increases the range of cancer-related epitopes presented to CD4+ helper T cells. Effective suppression of Ii protein was achieved with an antisense, phosphorothioate oligonucleotide, which was selected on the basis of (1) the RNase H activation assay, (2) an assay for Ii protein suppression, and (3) a test for potency with respect to the extent of base sequence ("sequence walking"). The SaI murine sarcoma, which is MHC-class-I+ and MHC-class-II-, Ii-protein-, upon transfection with genes for either interferon gamma or the MHC class II transactivator, came to express MHC class II molecules and Ii protein. In each line of transfected tumor cells, the antisense oligonucleotide profoundly suppressed Ii protein in 35%-55% cells, without affecting expression of MHC class II molecules. Inoculation of mice with such Ii-protein-suppressed tumor vaccine cells, after either formaldehyde fixation or X-irradiation, led to much greater protection against challenge with the parental SaI sarcoma than did inoculation with untreated cells. This approach to cancer cell vaccination can be applied in a wide range of human tumors.

Intracellular metabolism of a 2'-O-methyl-stabilized ribozyme after uptake by DOTAP transfection or asfree ribozyme. A study by capillary electrophoresis.

The uptake and cellular metabolism of a fluorescein-labelled synthetic ribozyme stabilized by 2'- O -methyl modification and a 3' inverted thymidine have been studied, employing capillary gel electrophoresis as a novel and efficient analytical method. After internalization by DOTAP transfection, electrophoretic peaks of intact ribozyme and different degradation products were easily resolved and the amount of intracellular intact ribozyme was quantified to >10(7) molecules/cell at the peak value after 4 h transfection. On further incubation the amount of intracellular intact ribozyme decreased due to both degradation and efflux from the cell. However, even after 48 h incubation there were still >10(6) intact ribozyme molecules/cell. Clear differences both in uptake and in metabolism were seen when comparing DOTAP transfection with the uptake of free ribozyme. Fluorescence microscopy studies indicated that the ribozyme was mainly localized in intracellular granules, probably not accessible to target mRNA. This implies that agents able to release the intact ribozyme from intracellular vesicles into the cytosol should have a considerable potential for increasing the biological effects of synthetic ribozymes.

In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions.

By truncating helix II to two base pairs in a hammerhead ribozyme having long flanking sequences (greater than 30 bases), the rate of cleavage in 1 mM magnesium can be increased roughly 100-fold. Replacing most of the nucleotides in a typical stem-loop II with 1-4 randomized nucleotides gave an RNA library that, even before selection, was more active in 1 mM magnesium than the parent ribozyme, but considerably less active than the truncated stem-loop II ribozyme. A novel, multiround selection for intermolecular cleavage was exploited to optimize this library for cleavage in low concentrations of magnesium. After three rounds of selection at sequentially lower concentrations of magnesium, the library cleaved substrate RNA 20-fold faster than the initial pool and was cloned. This pool was heavily enriched for one particular sequence (5'-CGUG-3') that represented 16 of 52 isolates (the next most common sequence was represented only six times). This sequence also represented the most active sequence, exceeding the activity of the short helix II variant under the conditions of the selection, thereby demonstrating the effectiveness of the selection technique. Analysis of the cleavage rates of RNAs made from eight isolates having different four-base insert sequences allowed assignment of highly preferred bases at each position in the insert. Analysis of pool clones having insert of differing lengths showed that, in general, activity decreased as the length of the insert decreased from 4 to 1. This supports the suggested role of stem-loop II in stabilizing the non-Watson-Crick interactions between the conserved bases of the catalytic core.

Fluorescence resonance energy transfer analysis of ribozyme kinetics reveals the mode of action of a facilitator oligonucleotide.

A defining characteristic of catalysts is the rate at which they can process multiple copies of substrate. In the case of synthetic hammerhead ribozymes that cleave an RNA sequence, binding of the ribozyme to the substrate and products is through base-paired duplexes. The kinetics of formation and dissociation of these duplexes can determine the turnover of the ribozyme. We have followed these processes in real time by using fluorescent labels that can interact through fluorescence resonance energy transfer (FRET). This approach has been used to identify the rate-limiting steps for a particular ribozyme and to reveal how turnover was improved by a facilitator oligonucleotide. It was found that dissociation of the ribozyme-substrate complex is faster than cleavage to products. Hence, to undergo cleavage, most substrate molecules must interact with a ribozyme more than once. In the presence of a facilitator oligonucleotide, the complex is stabilized so that cleavage is faster than dissociation. Under these circumstances, cleavage of the substrate becomes the most likely outcome following binding to the ribozyme.

Electron micrographic studies of transport of oligodeoxynucleotides across eukaryotic cell membranes.

Unmodified oligodeoxynucleotides (ODNs) were synthesized and tested for their ability to cross external eukaryotic cell membranes and to enter the cytosol and nucleus in tissue cultures. The ODNs were labeled with high-specific-activity [3H]thymidine (> or = 100 Ci/mmol), or [ alpha-32P]ATP or [ gamma-32P]ATP (300-1000 Ci/mmol; 1 Ci = 37 GBq), and the label was either in the central portion of the molecule or at the 3' or 5' end. The cells employed were for the most part 3T6 murine fibroblasts, grown in monolayers, either semiconfluent or confluent, but some experiments were carried out with chicken embryo fibroblasts or human HeLa cells. Parallel wells in the same experiment were prepared for electron microscopy or for cell fractionation and radioactivity assays. Electron microscopic autoradiography indicated that ODNs cross the external cell membrane, traverse the cytosol, and begin to enter the cell nucleus within a few seconds to 5 min at 37 degrees C in Dulbecco's medium without added serum. After 30-60 min of incubation with ODNs, abundant silver grains were observed at or just inside the nuclear membrane or well distributed across the nucleus, particularly in association with euchromatin. There was a paucity of silver grains associated with nucleoli. Cell entry of oligomer was related to cell cycling events and was energy dependent. Degradation of oligomer to monomers, with reincorporation into DNA, does not appear to explain these results. No sequestration of labeled oligomer in cytoplasmic vesicles en route from the exterior of the cell to the nucleus was observed. The observations are more suggestive of internalization of oligonucleotide by a mechanism as yet unclear or, alternatively, by a caveolar, potocytotic mechanism rather than by endocytosis.

DNA ligase I is associated with the 21 S complex of enzymes for DNA synthesis in HeLa cells.

Approximately 80% of the DNA ligase activity in HeLa cell extracts is associated with the 21 S enzyme complex that functions in simian virus 40 DNA replication in vitro (Malkas et al., Biochemistry 29, 6362-6374., 1990). The DNA ligase associated with the 21 S complex was purified extensively and its physical, enzymic and immunological properties characterized as DNA ligase I. The association of DNA ligase I with the 21 S complex of enzymes for DNA synthesis provides evidence for the physiological function of this DNA ligase in DNA replication in human cells.

Further studies on the use of oligonucleotide facilitators to increase ribozyme turnover.

We showed previously that the turnover of a hammerhead ribozyme cleaving a substrate RNA could be increased by an oligonucleotide (facilitator) that bound to the substrate continguously with the ribozyme. This phenomenon has been investigated further by varying the temperature and the number of base pairs formed between the ribozyme and substrate. The results support the hypothesis that facilitators act by cooperative binding and are beneficial when the rate of cleavage is limited by the stability of the ribozyme/substrate complex. The facilitator also promotes cleavage at lower concentrations of magnesium.

Enhancement of ribozyme catalytic activity by a contiguous oligodeoxynucleotide (facilitator) and by 2'-O-methylation.

RNA catalysts (ribozymes) designed to cleave sequences unique to viral RNA's might be developed as therapeutics. For this purpose, they would require high catalytic efficiency and resistance to nucleases. Reported here are two approaches that can be used in combination to improve these properties. First, catalytic efficiency can be improved by oligonucleotides (facilitators) that bind to the substrate contiguously with the 3'-end of the ribozyme. Second, 2'-O-methylation of flanking sequences of the ribozyme increases catalytic activity as well as resistance to nucleases. In combination with a facilitator oligodeoxynucleotide, the cleavage rate was increased 20 fold over that of the unmodified ribozyme.

Ribozymes that cleave an RNA sequence from human immunodeficiency virus: the effect of flanking sequence on rate.

Ribozymes designed to cleave sequences specific to viral RNA may be better antiviral agents than simple antisense oligonucleotides. High catalytic activity with the lowest possible chain length is desired for this purpose. We have synthesized several hammerhead ribozymes that cleave sequences from HIV-1 RNA. On reducing from 20 to 12 the base pairs formed with the substrate, the rate of cleavage at 37 degrees C increased 10-fold. Deletions from the stem/loop structure in the ribozyme also increased the initial rate of reaction.

The clearance and degradation of oligodeoxynucleotides following intravenous injection into rabbits.

A 32P-labeled oligodeoxynucleotide of 20 bases was found to be quite stable in freshly drawn rabbit or human blood and no degradation was apparent within 2 hr. In contrast, following injection into rabbits, the same oligodeoxynucleotide was cleared from the blood rapidly and at least 17% was completely degraded within 5 min. After 90 min, 16% of the oligonucleotide was found largely intact in the urine. At this time, radioisotope was also found in all the solid tissues tested but probably not as intact oligonucleotide.

Antisense antivirals.

The antiviral use of antisense oligonucleotides is described with particular reference to our own work on the inhibition of HIV-1. Subjects include the design, metabolism, and modification of these agents and the possible future use of ribozymes.