Facilitator oligonucleotides increase ribozyme RNA binding to full‐length RNA substrates in vitro

Denman, Robert B.

doi:10.1016/0014-5793(96)00125-1

Cited by 13 publications

(9 citation statements)

References 29 publications

(46 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3B). This suggests that PNA2 enhances DNAzyme activity by affecting the substrate binding step of the RNA cleavage reaction in cells, which was previously suggested by an in vitro study of hammerhead ribozyme [8]. Furthermore, T315IL DNAzyme combined with the facilitator PNA sensitized imatinib‐resistant leukemic cells to imatinib treatment with a significant reduction in cell viability (asterisked bar in Fig.…”

Section: Resultssupporting

confidence: 63%

“…1B). Previous attempts to improve ribozyme or DNAzyme activity have used facilitator oligonucleotides that bind RNA substrate at the termini of the ribozyme or DNAzyme [8–12]. Alternatively, biding arm of DNAzyme has been modified by incorporation of 2′‐ O ‐methyl RNA or locked nucleic acid (LNA) nucleoside analogues to enhance target affinity in vitro [13–16].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Site‐specific cleavage of mutant ABL mRNA by DNAzyme is facilitated by peptide nucleic acid binding to RNA substrate

et al. 2012

View full text Add to dashboard Cite

Edited by Tamas DalmayKeywords: DNAzyme Peptide nucleic acid (PNA) RNA cleavage Single nucleotide polymorphism (SNP) a b s t r a c t RNA-cleaving DNAzymes were constructed to target the point mutation in the BCR-ABL transcript that causes imatinib resistance in leukemic cells. We examined the effect of 12mer peptide nucleic acids (PNAs) as facilitator oligonucleotides that bind to RNA substrate at the termini of the DNAzyme to improve DNAzyme-mediated cleavage of full-length RNA. When imatinib-resistant cells were transfected with the facilitator PNA and DNAzyme, DNAzyme activity was enhanced and the cells were sensitized to imatinib treatment. Thus, facilitator PNA may be used to enhance activity of antisense oligonucleotide targeting the full-length transcript.

show abstract

Section: Resultssupporting

confidence: 63%

Section: Introductionmentioning

confidence: 99%

Site‐specific cleavage of mutant ABL mRNA by DNAzyme is facilitated by peptide nucleic acid binding to RNA substrate

et al. 2012

View full text Add to dashboard Cite

show abstract

“…Both in the studies reported here and in our earlier work (Goodchild, 1997), facilitator oligonucleotides have proven useful in many different examples for improving multiple-turnover cleavage by ribozymes having 5 or 6-base flanking sequences. Other workers have also used facilitator oligonucleotides with different ribozymes (Denman, 1996;Jankowsky and Schwenzer, 1996;Jankowsky et al, 1997). Present studies showed that considerable improvements could be made in ribozyme-catalyzed multiple-turnover cleavage by optimizing flanking sequences and assisting hybridization using facilitator oligonucleotides.…”

Section: Modified Ribozymes 73mentioning

confidence: 89%

“…The facilitator oligonucleotide stabilizes the complex between the ribozyme and substrate, thereby increasing the likelihood it will undergo cleavage before it dissociates (Perkins et al, 1996). The effect of facilitator oligonucleotides is greater with longer substrates, and an additional benefit may be in assisting binding or ribozymes to sites that are partially occluded by secondary structure in the substrate (Denman, 1996;Jankowsky and Schwenzer, 1996;Jankowsky et al, 1997). This may result from the smaller molecular bulk of facilitator oligonucleotides compared with hammerhead ribozymes.…”

Section: Figmentioning

confidence: 95%

Optimization of Hammerhead Ribozymes for the Cleavage of S100A4 (CAPL) mRNA

Hovig

Mælandsmo

Fodstad

et al. 2001

Antisense and Nucleic Acid Drug Development

View full text Add to dashboard Cite

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.

show abstract

“…Such facilitators are oligonucleotides that are capable of binding to the substrate adjacent to the termini of the hammerhead ribozyme. They enhance the turnover of short RNA substrates (Goodchild, 1992;Nesbitt and Goodchild, 1994;Jankowsky and Schwenzer, 1996a;Perkins et al, 1996) as well as the cleavage of long RNA substrates (Denman, 1993;Denman 1996;Jankowsky and Schwenzer, 1996b;Jankowsky et al, 1997;Jankowsky and Schwenzer, 1998).…”

Section: Introductionmentioning

confidence: 99%

Oligonucleotide Facilitators Enhance the Catalytic Activity of RNA-Cleaving DNA Enzymes

Horn

Schwenzer²

1999

Antisense and Nucleic Acid Drug Development

View full text Add to dashboard Cite

From in vitro selection studies, DNA structures have been found that cleave target RNA sequence specifically and show a certain similarity to the well-investigated hammerhead ribozymes. Such DNA enzymes are more resistant to nuclease-mediated degradation than RNA enzymes. On the other hand, their cleavage activity is lower than the activity of hammerhead ribozymes. In the present study, we improved the activity of DNA enzymes by adding oligonucleotide facilitators complementary to the 5' and the 3' ends of the substrate to the cleavage reaction. DNA enzyme activity in vitro was monitored under multiple turnover conditions using short RNA model substrates. We have shown that oligonucleotide facilitators strongly enhance the multiple turnover activity of the DNA enzyme reaction. In one of our model systems with a suitable facilitator combination, we were able to observe a more than 200-fold enhancement of the k(cat)/Km value. The comparison of two DNA enzyme-substrate systems showed that the principal effects of the facilitators were independent of the substrate sequence. However, the degree of facilitator effect was noticeably dependent on the basic catalytic efficiency of DNA enzymes. Furthermore, the efficiency of the DNA enzyme reaction with facilitator was compared with the reaction of a DNA enzyme with a stem sequence extended by the sequence of the facilitator. The multiple turnover activity of such a "long DNA enzyme" is higher than the activity of the short DNA enzyme without facilitators. However, when compared with the multiple turnover reactions of the short DNA enzyme with facilitator, the reaction with the long DNA enzyme is considerably slower. The results obtained with our model systems demonstrate that oligonucleotide facilitators enable DNA enzymes to act as effective multiple turnover catalysts by cleavage of RNA substrates.

show abstract

Facilitator oligonucleotides increase ribozyme RNA binding to full‐length RNA substrates in vitro

Cited by 13 publications

References 29 publications

Site‐specific cleavage of mutant ABL mRNA by DNAzyme is facilitated by peptide nucleic acid binding to RNA substrate

Site‐specific cleavage of mutant ABL mRNA by DNAzyme is facilitated by peptide nucleic acid binding to RNA substrate

Optimization of Hammerhead Ribozymes for the Cleavage of S100A4 (CAPL) mRNA

Oligonucleotide Facilitators Enhance the Catalytic Activity of RNA-Cleaving DNA Enzymes

Contact Info

Product

Resources

About