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A Siwkowski

Publications and source records attributed to A Siwkowski.

10 recordsLinked to original sources
PubMed1999

The sCYMV1 hairpin ribozyme: targeting rules and cleavage of heterologous RNA.

The catalytic center of the RNA from the negative strand of the satellite RNA of chicory yellow mottle virus type 1 (sCYMV1) is in the hairpin ribozyme family, has catalytic activity, and cleaves substrates before a preferred GUA sequence. This is different from that of the satellite RNA from the negative strand of tobacco ringspot virus (sTRSV) which prefers a GUC sequence at the site of cleavage. The sCYMV1 hairpin ribozyme has now been developed for cleaving heterologous RNA substrates. When helix 1 was extended from the native 5 bp to 6 bp with a newly added A:U base pair, catalytic activity increased three-fold. The preferred sequence for the substrate loop was the native A*GUA sequence where * is the site of cleavage. When each nucleotide in this sequence was changed to each of the other three nucleotides, catalytic activity decreased 66-100%. RNA targets, containing this A*GUA sequence, were located in both human papillomavirus and HIV-1. Ribozymes were developed which efficiently cleaved these targets in vitro. These results identify a new class of hairpin ribozymes capable of cleaving substrates before a preferred GUA sequence rather than the GUC preferred by the sTRSV hairpin ribozyme. This expands the repertoire of target sites available for gene therapy using the hairpin ribozyme.

Gene Targeting↗
PubMed1998

Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes.

HPV-16 E6 and E7 genes are required to efficiently immortalize a broad spectrum of cell types including cervical keratinocytes. Therefore, the E6/E7 genes can be considered relevant targets for anti-cancer therapy. We produced several engineered hairpin (HP) ribozymes to specifically disrupt HPV-16 E6/E7 mRNA. After extensive biochemical characterization, one anti-E6 HP ribozyme (R434) was selected for in vivo testing because of its superior catalytic capabilities. When expressed in cis, R434 efficiently inhibited E6 in vitro translation. Cis-expression of the HP ribozyme with HPV-16 E6/E7 genes in normal human keratinocytes reduced the growth rate and prevented immortalization. RNA analysis by reverse transcription-PCR showed that E6/E7 transcripts were cleaved in post-transfected cells and virtually were eliminated after long term expression. Of interest, an inactive version of the HP also was able to significantly affect the immortalizing ability of E6/E7, probably through passive hybridization. The combination of passive and cleaving antisense RNA therefore is established as an effective inhibitor of HPV-16 E6/E7 immortalization.

Antiviral Agents↗
PubMed1998

Mutational analysis of loops 1 and 5 of the hairpin ribozyme.

A comprehensive analysis of base preferences for all positions in loops 1 and 5 of the hairpin ribozyme-substrate complex was carried out using a cis-ribozyme tethered to substrate by a pentapyrimidine loop. Ribozyme-substrate molecules were mutated to contain each of the three non-native base variations at each of the eight positions within these loops. Catalytic activity was measured for each mutant and compared to the activity of the original native sequence. This was the first time all base positions in these loops have been mutated to all variants and kinetically characterized. Various effects were found, ranging from invariant base positions to those with nearly complete tolerance of any base change. Two positions resulted in cleavage rates below the lower limit of accurate quantification for all non-wild-type base substitutions. These positions are G8 in the ribozyme and Gs6 in the substrate. When A10 was substituted with a pyrimidine, self-cleavage activity fell below the lower limit of detection while the remaining positions showed varying base preferences. The information reported here on loops 1 and 5 combined with previous mutagenesis data on loops 2 and 4 [Siwkowski, A., Shippy, R., and Hampel, A. (1997) Biochemistry 36, 3930-3940] completed a comprehensive mutational/kinetic analysis of every base position located within all the required loops of the hairpin ribozyme-substrate complex and allowed for the development of a mechanism for catalysis which is proposed.

Amino Acid Sequence↗
PubMed1998

Screening for important base identities in the hairpin ribozyme by in vitro selection for cleavage.

Random mutagenesis followed by an in vitro selection procedure was shown to be capable of identifying important bases of the hairpin ribozyme for cleavage of an RNA target sequence. The selection scheme enriched the RNA population for those molecules capable of efficient site-specific self-cleavage in the absence of ligation. Cleavable mutants were selected for all positions in loop 4 except for position A38, supporting the notion that A38 is an important base in the hairpin ribozyme. This has been confirmed by direct mutagenesis, validating the utility of this procedure. Thus, the method developed and reported here has utility for the selection of efficient hairpin ribozymes capable of highly efficient cleavage of a substrate RNA without a requirement for ribozyme-catalyzed ligation, conditions desired for many applications of catalytic RNA such as gene therapy.

Base Composition↗
PubMed1997

Analysis of hairpin ribozyme base mutations in loops 2 and 4 and their effects on cis-cleavage in vitro.

In order to determine base requirements in loops 2 and 4 of the hairpin ribozyme, a comprehensive mutational analysis of the wild type sequence was done. Each base position in these two loops was mutated to contain each of the three non-wild type bases, and the effects of these mutations were analyzed using cis-cleavage assays. The method of data analysis allowed for the determination of self-cleavage rates as well as the fraction of transcripts produced which were uncleavable. Three positions in loop 2 (A22, A23, and C25) and one position in loop 4 (A38) resulted in no detectable self-cleavage when mutated to any of the non-wild type bases. The remainder of the base positions showed varying degrees of tolerance to base mutations with respect to their support of cis-cleavage. Evidence was obtained for the presence of a non-Watson-Crick base pair between A26 and G36 in the catalytic conformation of the hairpin ribozyme. On the basis of these results, a two-dimensional model for the hairpin ribozyme is presented.

Base Composition↗
PubMed1997

Design of the hairpin ribozyme for targeting specific RNA sequences.

The following steps should be taken when designing the hairpin ribozyme to cleave a specific target sequence: 1. Select a target sequence containing BN*GUC where B is C, G, or U. 2. Select the target sequence in areas least likely to have extensive interfering structure. 3. Design the conventional hairpin ribozyme as shown in Fig. 1, such that it can form a 4 bp helix 2 and helix 1 lengths up to 10 bp. 4. Synthesize this ribozyme from single-stranded DNA templates with a double-stranded T7 promoter. 5. Prepare a series of short substrates capable of forming a range of helix 1 lengths of 5-10 bp. 6. Identify these by direct RNA sequencing. 7. Assay the extent of cleavage of each substrate to identify the optimal length of helix 1. 8. Prepare the hairpin tetraloop ribozyme to determine if catalytic efficiency can be improved.

Base Sequence↗
PubMed1994

Alteration of hairpin ribozyme specificity utilizing PCR.

We have developed a method by which a researcher can quickly alter the specificity of a trans hairpin ribozyme. Utilizing this PCR method, two oligonucleotides, and any target vector, new ribozyme template sequences can be generated without the synthesis of longer oligonucleotides. We have produced templates with altered specificity for both standard and modified (larger) ribozymes. After transcription, these ribozymes show specific cleavage activity with the new substrate beta-glucuronidase (GUS), and no activity against the original substrate (HIV-1, 5' leader sequence). Utilizing this technique, it is also possible to produce an inactive ribozyme that can be used as an antisense control. Applications of this procedure would provide a rapid and economical system for the assessment of trans ribozyme activity.

Base Sequence↗