Ribozyme mediated cleavage of acute phase serum amyloid A (A‐SAA) mRNA in vitro

Gaughan, Derval J.; Steel, D. M.; Whitehead, Alexander S.

doi:10.1016/0014-5793(95)01118-x

Cited by 7 publications

(4 citation statements)

References 19 publications

(18 reference statements)

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“…This cleavage was greatly influenced by the concentration of MgCl 2 and almost complete cleavage was obtained at 100 mM MgCl 2 . Similar observations have been made for other unrelated ribozymes earlier [28]. A slight increase in the extent of cleavage was observed with the addition of a denaturing step before adding the MgCl 2 for initiating the cleavage reaction.…”

Section: Discussionsupporting

confidence: 84%

Sequence specific cleavage of the HIV‐1 coreceptor CCR5 gene by a hammer‐head ribozyme and a DNA‐enzyme: inhibition of the coreceptor function by DNA‐enzyme

Goila¹,

Banerjea²

1998

FEBS Letters

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The chemokine receptor CCR5 is used as a major coreceptor for fusion and entry by non-syncytia inducing macrophage tropic isolates of HIV-1, which is mainly involved in transmission. Individuals who are homozygous for the v v32 allele of CCR5 are usually resistant to HIV-1 infection and continue to lead a normal healthy life. Thus this gene is dispensable and is, therefore, an attractive target in the host cell for interfering specifically with the virus-host interaction. With the aim to develop a specific antiviral approach at the molecular level, we have synthesized a hammer-head ribozyme and a DNAenzyme. Both ribozyme and DNA-enzyme cleaved the CCR5 RNA in a sequence specific manner. This cleavage was protein independent but Mg P dependent. The extent of cleavage increased with increasing concentration of magnesium chloride. DNA-enzyme was more effective in cleaving a full length (1376 bases) in vitro generated transcript of CCR5 gene. In this communication, we show that the DNA-enzyme when introduced into a mammalian cell, results in decreased CD4-CCR5-gp160 mediated fusion of cell membranes. Potential applications of these trans acting molecules are discussed.z 1998 Federation of European Biochemical Societies.

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Section: Discussionsupporting

confidence: 84%

Sequence specific cleavage of the HIV‐1 coreceptor CCR5 gene by a hammer‐head ribozyme and a DNA‐enzyme: inhibition of the coreceptor function by DNA‐enzyme

Goila¹,

Banerjea²

1998

FEBS Letters

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“…Because of their catalytic nature, ribozymes are considered to be more effective than antisense reagents; as the latter act stoichiometrically, very high levels would be required to have a significant impact on protein production from a massively induced gene such as that encoding A-SAA2. The design, synthesis and cleavage capacity of a hammerhead ribozyme targeted against human A-SAA2 mRNA has been described previously [25]. In this paper we report the generation of a hammerhead ribozyme targeted against mouse A-SAA2 mRNA and demonstrate its long-term cleavage capacity.…”

Section: Introductionmentioning

confidence: 72%

“…We previously used the RNAPlotFold program to predict the secondary structure of human A-SAA2 mRNA so that the most accessible GUC target site for an anti human A-SAA2 ribozyme could be identified. A target site carried by an open-loop region at the end of a long stem was chosen and effective cleavage of the substrate at this site was demonstrated [25]. This same strategy was used for choosing the target site for a ribozyme directed against mouse A-SAA2 mRNA.…”

Section: Resultsmentioning

confidence: 99%

“…Computer-generated predictions of mRNA secondary structures have been used to identify the most accessible ribozyme target site on mRNA substrates [25,29,31]. We previously used the RNAPlotFold program to predict the secondary structure of human A-SAA2 mRNA so that the most accessible GUC target site for an anti human A-SAA2 ribozyme could be identified.…”

Section: Resultsmentioning

confidence: 99%

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Efficient In Vitro Cleavage of Mouse Acute Phase Serum Amyloid A mRNA Mediated by a Synthetic Hammerhead Ribozyme

Gaughan

Whitehead

1997

Scand J Immunol

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Gaughan DJ, Whitehead AS. Efficient In Vitro Cleavage of Mouse Acute Phase Serum Amyloid A mRNA Mediated by a Synthetic Hammerhead Ribozyme. Scand J Immunol 1997;46:51-58 The authors designed a hammerhead ribozyme, mRibSAA2, to cleave the mRNA for mouse acute phase serum amyloid A 2 (A-SAA2), a major acute phase protein that is massively induced during inflammation and that is deposited as fibrils during secondary amyloidosis. Using computer based secondary structure analysis, a GUC triplet (nucleotides 408-410) on a predicted stem loop in A-SAA2 mRNA was chosen as the target site for mRibSAA2. The ribozyme was tested in vitro and gave efficient and specific magnesium-dependent cleavage of mouse A-SAA2 mRNA into the expected fragments of 197 and 425 bases. The authors also demonstrated that the ribozyme retains cleavage activity over several hours. The use of the mRibSAA2 ribozyme as a research tool and possible therapeutic agent in mouse models of amyloidosis is discussed.

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Nucleic Acid-Mediated Cleavage of M1 Gene of Influenza A Virus Is Significantly Augmented by Antisense Molecules Targeted to Hybridize Close to the Cleavage Site

et al. 2011

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Influenza A virus genome segment 7 encodes protein M1, which is the matrix protein playing crucial role in the virus life cycle. Any antiviral strategy that aims at reducing, in particular, the expression of this genome segment should, in principle, reduce the infectivity of the virus. We developed a specific antiviral approach at the molecular level and designed several novel 10-23 DNAzymes (Dz) and hammerhead ribozymes (Rz), specifically targeted to cleave at the conserved domains of the influenza virus M1 RNA. We sought to use antisense molecules with the hope that it will facilitate the ribozyme-mediated cleavage. We observed that the Mg(2+)-dependent sequence-specific cleavage of M1 RNA was achieved by both the Dz and Rz in a dose-dependent manner. This combination of catalytic Dz and Rz with antisense molecules, in principle, resulted in more effective gene suppression, inhibited the whole virus replication in host cell, and thus could be exploited for therapeutic purposes.

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Ribozyme mediated cleavage of acute phase serum amyloid A (A‐SAA) mRNA in vitro

Cited by 7 publications

References 19 publications

Sequence specific cleavage of the HIV‐1 coreceptor CCR5 gene by a hammer‐head ribozyme and a DNA‐enzyme: inhibition of the coreceptor function by DNA‐enzyme

Sequence specific cleavage of the HIV‐1 coreceptor CCR5 gene by a hammer‐head ribozyme and a DNA‐enzyme: inhibition of the coreceptor function by DNA‐enzyme

Efficient In Vitro Cleavage of Mouse Acute Phase Serum Amyloid A mRNA Mediated by a Synthetic Hammerhead Ribozyme

Nucleic Acid-Mediated Cleavage of M1 Gene of Influenza A Virus Is Significantly Augmented by Antisense Molecules Targeted to Hybridize Close to the Cleavage Site

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