Hammerhead ribozymes are attractive tools in antisense gene inactivation because of their catalytic cleavage of target molecules. High sequence discrimination should be possible, since the cleavage efficiencies were already significantly reduced, if single base changes in substrate RNA introduce mismatches next to the cleavage site. This was observed at the first innermost base pair in helix I and the two innermost base pairs in helix III. In addition to its position, the nature of the mismatch pair was important.

Autoradiography↗

PubMed1996

Differences in the interaction of Escherichia coli RNase P RNA with tRNAs containing a short or a long extra arm.

The phosphorothioate footprinting technique was applied to the investigation of phosphate moieties in tRNA substrates involved in interactions with M1 RNA, the catalytic subunit of Escherichia coli RNase P. In general agreement with previous data, all affected sites were localized in acceptor stem and T arm. But the analyzed examples for class I (Saccharomyces cerevisiae pre-tRNA(Phe) with short variable arm) and class II tRNAs (E. coli pre-tRNA(Tyr) with large variable arm) revealed substantial differences. In the complex with pre-tRNA(Phe), protection was observed at U55, C56, and G57, along the top of the T loop in the tertiary structure, whereas in pre-tRNA(Tyr), the protected positions were G57, A58, and A59, at the bottom of the T loop. These differences suggest that the size of the variable arm affects the spatial arrangement of the T arm, providing a possible explanation for the discrepancy in reports about the D arm requirement in truncated tRNA substrates for eukaryotic RNase P enzymes. Enhanced reactivities were found near the junction of acceptor and T stem (U6, 7, 8 in pre-tRNA(Phe) and G7, U63, U64 in pre-tRNA(Tyr)). This indicates a partial unfolding of the tRNA structure upon complex formation with RNase P RNA.

Base Sequence↗

PubMed1995

Similar cleavage efficiencies of an oligoribonucleotide substrate and an mdr1 mRNA segment by a hammerhead ribozyme.

Hammerhead ribozymes (Rz) can specifically recognize and cleave target RNAs in trans. This makes them attractive in antisense RNA approaches for specific gene inactivation in vivo. A severe limitation is the poor cleavage efficiency of large RNA substrates, in contrast to the high activities observed with small oligoribonucleotides (oligos) as model substrates. It was suggested that the low efficiency is caused by poor accessibility of the target sequence in the structure of the long RNA substrates. This means it should be possible to overcome this limitation by judicious choice of the target sequence, although experimental proof was lacking. We observed similar cleavage efficiencies of small and large RNA substrates with a hammerhead Rz directed against multidrug resistance-encoding mdr1 mRNA. Accordingly, large RNAs can also be good substrates, if an optimal target sequence is selected.

Base Sequence↗

PubMed1995

Enzymatic synthesis of 2'-modified nucleic acids: identification of important phosphate and ribose moieties in RNase P substrates.

For the first time mosaic nucleic acids composed of 50% RNA and 50% DNA can be obtained as transcripts with T7 RNA polymerase. Two NTPs could be replaced simultaneously in a transcription reaction. This means more than 40 deoxynucleotides were inserted in one transcript. Previously, a maximum of two deoxynucleotides could be incorporated and 2'-O-methyl-NTPs were not substrates at all. We obtained reasonable transcript yields with a maximal level of 99% 2'-O-methyl-NTPs, and the products contained up to 58% 2'-O-methylnucleotides at more than 20 positions. Sequence-specific nucleotide incorporation was monitored by sequence ladders (partial alkali or iodine cleavage). No base misincorporations were detected with 100% dGTP, dCTP and dTTP, and with partial incorporation of dATP alpha S, 2'-O-methyl-GTP alpha S and 2'-O-methyl-CTP alpha S, whereas they were found with dATP, 2'-O-methyl-ATP alpha S and 2'-O-methyl-UTP alpha S. Quantitative data allow predetermined modification levels of partially modified transcripts. Highly modified transcripts can be used for structural and functional studies, in modification interference approaches and for in vitro evolution procedures. Modification interference studies revealed a small number of important phosphate and ribose moieties in RNase P substrates. The conversion of T7 RNA polymerase to a DNA polymerase extends the observation that there is no absolute distinction between RNA and DNA polymerases. Accordingly, an adapted concept of a primordial RNA world is presented.

Base Sequence↗

PubMed1995

Tumefactive megalocytic interstitial nephritis in a patient with Escherichia coli bacteremia.

Megalocytic interstitial nephritis is rare and primarily affects the cortex in an otherwise normal kidney. We recently encountered a patient with Escherichia coli bacteremia and oliguric acute renal failure who died of gram-negative septicemia. At autopsy, this patient's kidneys displayed typical features of megalocytic interstitial nephritis. We were able to perform special stains suggesting that the histiocytic interstitial cells originated from infiltrating macrophages. Our patient illustrates that macrophage proliferation can result in interstitial inflammation sufficiently severe to cause anuric acute renal failure.

Acute Kidney Injury↗

PubMed1995

Towards a new concept of gene inactivation: specific RNA cleavage by endogenous ribonuclease P.

In the first part of this chapter, general concepts for gene inactivation, antisense techniques and catalytic RNAs (ribozymes) are presented. The requirements for modified oligonucleotides are discussed with their effects on the stability of base-paired hybrids and on resistance against nuclease attack. This also includes the problems in the choice of an optimal target sequence within the inactivated RNA and the options of cellular delivery systems. The second part describes the recently introduced antisense concept based on the ubiquitous cellular enzyme ribonuclease P. This system is unique, since the substrate recognition requires the proper tertiary structure of the cleaved RNA. General properties and possible advantages of this approach are discussed.

Animals↗

PubMed1993

Efficient cleavage of pre-tRNAs by E. coli RNAse P RNA requires the 2'-hydroxyl of the ribose at the cleavage site.

RNAse P cleaves pre-tRNAs to liberate 5'-flanks and 5'-matured, 5'-phosphorylated tRNAs. It is not evident if the 2'-hydroxyls of the ribose moieties in the substrate are involved in the reaction. To study their influence in two different pre-tRNAs, we have modified specifically the 2'-hydroxyl groups at the cleavage site and in neighbouring positions. We have shown that these hydroxyls are important but not essential for the processing of these substrates by E. coli RNase P RNA (M1 RNA). The reduction in the catalytic efficiency was moderate for 2'-deoxy and severe for 2'-methoxy substitutions at the cleavage site. Additional effects of modifications in neighbouring positions were smaller. Based on our data we suggest that the modifications do not interfere with binding of the substrate, whereas they prevent an optimal steric arrangement for the hydrolysis reaction.

Base Sequence↗

PubMed1993

Modification interference approach to detect ribose moieties important for the optimal activity of a ribozyme.

A new approach for modification interference studies is presented. It involves the use of phosphorothioates as a handle to analyze any desired base or sugar modification. This method was applied to identify ribose and phosphate moieties which could be important in the pre-tRNA recognition of E. coli RNase P RNA (M1 RNA). The utility of this technique was confirmed by detecting the inhibitory effect of a deoxyribose in the 5'-flank (position-1). This site was already known to interfere with RNase P cleavage, if modified. We have analyzed pre-tRNA(Tyr) and pre-tRNA(Phe) and found different interference patterns for both tRNAs. Two unpaired regions were involved in both pre-tRNAs. Phosphorothioates interfered at the transition between acceptor- and D-arms. The results with deoxythymidines in the T-loop indicated that deoxyribose moieties or the extra methyl group in thymidine could interfere with RNAse P cleavage. These data suggest that even in complete pre-tRNAs, only a few intact ribonucleotides are important in the substrate recognition by RNase P. We have demonstrated the potential of this new approach which offers many future applications in all fields involving nucleic acids, for example RNA processing, action of ribozymes, tRNA charging and studies related to DNA promoter recognition.

Base Sequence↗

PubMed1993

Enzymatic RNA synthesis with deoxynucleoside 5'-O-(1-thiotriphosphates).

We have investigated the incorporation of 2'-deoxynucleoside-5'-O-(1-thiotriphosphates) into RNA transcripts using T7 RNA polymerase. With the exception of [alpha-S]dGTP, we obtained full-length transcripts of pre-tRNA(Phe) and pre-tRNA(Tyr) using an appropriate mixture of 2'-deoxynucleoside 5'-O-(1-thiotriphosphate) and the corresponding normal nucleoside triphosphate. The yields of the transcripts were comparable to those obtained with unmodified NTPs. Both substrates, [alpha-S]dTTP and [alpha-S]dATP, were inserted specifically. However, [alpha-S]dCTP was excluded at specific sites. We could not obtain transcripts using the deoxyguanosine derivative.

Base Sequence↗

PubMed1993

Antisense oligoribonucleotides and RNase P. A great potential.

This paper presents the possible--but at present mostly hypothetical--applications of RNase P for the specific inactivation of target RNAs. The natural substrates for RNase P are pre-tRNAs. The enzyme can also recognize and cleave smaller model substrates. These are simple hairpins for bacterial RNase P whereas the requirements for eukaryotic RNase P are more complex and less well understood. It is possible to split the RNase P substrates into two separate RNA molecules. One part of the split substrate RNA contains the RNase P cleavage site and the 5'-terminal half of the acceptor stem, embedded in a large target RNA. The other part of the substrate RNA provides the 3'-terminal half of the acceptor stem and it serves as antisense sequence ('external guide sequence'). Both parts can hybridize and reconstitute a functional substrate for RNase P; this results in cleavage of the target RNA by RNase P. The properties of this system are presented and advantages and problems discussed.

Animals↗

PubMed1993

Phosphorothioates in pre-tRNAs can change the specificities of RNAses P or reduce the cleavage efficiencies.

Several phosphorothioate-modified E coli and yeast pre-tRNAs were synthesized. If this modification included the phosphodiester at the RNase P cleavage site, two different effects were observed. With some pre-tRNAs the RNase P cleavage efficiency was severely reduced, whereas with other pre-tRNAs a new reaction type for RNase P was observed. Unlike the previously studied base or ribose modifications, phosphorothioates resulted in aberrant cleavages at unmodified phosphodiesters. These new sites could be located in the 5'-flank or in the acceptor stem of the tRNA domain. Modified mutants of E coli pre-tRNA(Tyr) with different base pairs at the RNase P cleavage site were cleaved with reduced efficiencies, but no aberrant products were observed.

Base Composition↗

PubMed1992

A solid phase method for mapping restriction sites.

Explore the source record for details and available documents.

DNA, Bacterial↗

PubMed1992

Initiator oligonucleotides for the combination of chemical and enzymatic RNA synthesis.

Transcription reactions with T7 RNA polymerase were performed in the presence of short oligonucleotides (oligos) with guanosine at the 3'-end. We obtained transcripts which had included these 'initiator oligos' at their 5'-termini. The oligos could contain mixtures of deoxyribo-, ribo-, 2'-O-methylated and biotinylated nucleotides. Only the 3'-terminal guanosine of these oligos was encoded in the template DNA at the transcription start point, in contrast to the remainder of the sequence. This 5'-terminal sequence is variable and eliminates the limitation that transcripts must start with a 5'-terminal guanosine. With a 5'-biotinylated dinucleotide, we obtained end-labeled RNAs suitable for nonradioactive RNA sequencing.

Biotin↗