Cleavage of full-length beta APP mRNA by hammerhead ribozymes.
Identifieur interne : 002913 ( PubMed/Corpus ); précédent : 002912; suivant : 002914Cleavage of full-length beta APP mRNA by hammerhead ribozymes.
Auteurs : R B DenmanSource :
- Nucleic acids research [ 0305-1048 ] ; 1993.
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : Amyloid beta-Protein Precursor.
- chemical , metabolism : RNA, Catalytic, RNA, Messenger.
- Base Sequence, Kinetics, Molecular Sequence Data, Nucleic Acid Conformation, Substrate Specificity.
Abstract
The sequences surrounding the first 5'GUC3' in the mRNA encoding the Alzheimer amyloid peptide precursor (beta APP) were used to construct a pair of transacting hammerhead ribozymes. Each ribozyme contained the conserved core bases of the hammerhead motif found in the positive strand of satellite RNA of tobacco ringspot virus [(+)sTRSV] and two stems, 7 and 8 bases long, complementary to the target, beta APP mRNA. However, one of the ribozyme cleaving strands was lengthened at its 3' end to include the early splicing and polyadenylation signal sequences of SV40 viral RNA. This RNA, therefore, more closely mimics transcripts produced by RNA polymerase II from eucaryotic expression vectors in vivo. RNA, prepared by run-off transcription of cDNA oligonucleotide or plasmid constructs containing a T7 RNA polymerase promoter was used to characterize several properties of the cleavage reaction. In the presence of both ribozyme cleaving strands magnesium-ion dependent cleavage of a model 26 base beta APP substrate RNA or full-length beta APP-751 mRNA was observed at the hammerhead consensus cleavage site. Neither ribozyme was active against non-message homologs of beta APP mRNA, nor was cleavage detected when point mutations were made in the conserved core sequences. However, the kcat/Km at 37 degrees C in 10 mM Mg+2 of the longer ribozyme was reduced twenty-fold when model and full-length substrates were compared. The use of short deoxyoligonucleotides (13-17 mers) that bind upstream of the ribozyme was found to enhance the rate of cleavage of the full-length but not beta APP model substrate RNAs. The rate of enhancement depended on both the length of the deoxyoligonucleotide used as well as its site of binding with respect to the ribozyme. These data demonstrate the utility of ribozymes to cleave target RNAs in a catalytic, site-specific fashion in vitro. Direct comparison of the efficiency of different ribozyme constructs and different modulating activities provide an experimental strategy for designing more effective ribozymes for therapeutic purposes.
DOI: 10.1093/nar/21.17.4119
PubMed: 8371986
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Cleavage of full-length beta APP mRNA by hammerhead ribozymes.</title><author><name sortKey="Denman, R B" sort="Denman, R B" uniqKey="Denman R" first="R B" last="Denman">R B Denman</name><affiliation><nlm:affiliation>New York Institute for Basic Research in Developmental Disabilities, Staten Island 10314.</nlm:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="1993">1993</date><idno type="RBID">pubmed:8371986</idno><idno type="pmid">8371986</idno><idno type="doi">10.1093/nar/21.17.4119</idno><idno type="wicri:Area/PubMed/Corpus">002913</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">002913</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">Cleavage of full-length beta APP mRNA by hammerhead ribozymes.</title><author><name sortKey="Denman, R B" sort="Denman, R B" uniqKey="Denman R" first="R B" last="Denman">R B Denman</name><affiliation><nlm:affiliation>New York Institute for Basic Research in Developmental Disabilities, Staten Island 10314.</nlm:affiliation></affiliation></author></analytic><series><title level="j">Nucleic acids research</title><idno type="ISSN">0305-1048</idno><imprint><date when="1993" type="published">1993</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Amyloid beta-Protein Precursor (genetics)</term><term>Base Sequence</term><term>Kinetics</term><term>Molecular Sequence Data</term><term>Nucleic Acid Conformation</term><term>RNA, Catalytic (metabolism)</term><term>RNA, Messenger (metabolism)</term><term>Substrate Specificity</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Amyloid beta-Protein Precursor</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>RNA, Catalytic</term><term>RNA, Messenger</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Base Sequence</term><term>Kinetics</term><term>Molecular Sequence Data</term><term>Nucleic Acid Conformation</term><term>Substrate Specificity</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The sequences surrounding the first 5'GUC3' in the mRNA encoding the Alzheimer amyloid peptide precursor (beta APP) were used to construct a pair of transacting hammerhead ribozymes. Each ribozyme contained the conserved core bases of the hammerhead motif found in the positive strand of satellite RNA of tobacco ringspot virus [(+)sTRSV] and two stems, 7 and 8 bases long, complementary to the target, beta APP mRNA. However, one of the ribozyme cleaving strands was lengthened at its 3' end to include the early splicing and polyadenylation signal sequences of SV40 viral RNA. This RNA, therefore, more closely mimics transcripts produced by RNA polymerase II from eucaryotic expression vectors in vivo. RNA, prepared by run-off transcription of cDNA oligonucleotide or plasmid constructs containing a T7 RNA polymerase promoter was used to characterize several properties of the cleavage reaction. In the presence of both ribozyme cleaving strands magnesium-ion dependent cleavage of a model 26 base beta APP substrate RNA or full-length beta APP-751 mRNA was observed at the hammerhead consensus cleavage site. Neither ribozyme was active against non-message homologs of beta APP mRNA, nor was cleavage detected when point mutations were made in the conserved core sequences. However, the kcat/Km at 37 degrees C in 10 mM Mg+2 of the longer ribozyme was reduced twenty-fold when model and full-length substrates were compared. The use of short deoxyoligonucleotides (13-17 mers) that bind upstream of the ribozyme was found to enhance the rate of cleavage of the full-length but not beta APP model substrate RNAs. The rate of enhancement depended on both the length of the deoxyoligonucleotide used as well as its site of binding with respect to the ribozyme. These data demonstrate the utility of ribozymes to cleave target RNAs in a catalytic, site-specific fashion in vitro. Direct comparison of the efficiency of different ribozyme constructs and different modulating activities provide an experimental strategy for designing more effective ribozymes for therapeutic purposes.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">8371986</PMID><DateCompleted><Year>1993</Year><Month>10</Month><Day>14</Day></DateCompleted><DateRevised><Year>2019</Year><Month>05</Month><Day>01</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Print">0305-1048</ISSN><JournalIssue CitedMedium="Print"><Volume>21</Volume><Issue>17</Issue><PubDate><Year>1993</Year><Month>Aug</Month><Day>25</Day></PubDate></JournalIssue><Title>Nucleic acids research</Title><ISOAbbreviation>Nucleic Acids Res.</ISOAbbreviation></Journal><ArticleTitle>Cleavage of full-length beta APP mRNA by hammerhead ribozymes.</ArticleTitle><Pagination><MedlinePgn>4119-25</MedlinePgn></Pagination><Abstract><AbstractText>The sequences surrounding the first 5'GUC3' in the mRNA encoding the Alzheimer amyloid peptide precursor (beta APP) were used to construct a pair of transacting hammerhead ribozymes. Each ribozyme contained the conserved core bases of the hammerhead motif found in the positive strand of satellite RNA of tobacco ringspot virus [(+)sTRSV] and two stems, 7 and 8 bases long, complementary to the target, beta APP mRNA. However, one of the ribozyme cleaving strands was lengthened at its 3' end to include the early splicing and polyadenylation signal sequences of SV40 viral RNA. This RNA, therefore, more closely mimics transcripts produced by RNA polymerase II from eucaryotic expression vectors in vivo. RNA, prepared by run-off transcription of cDNA oligonucleotide or plasmid constructs containing a T7 RNA polymerase promoter was used to characterize several properties of the cleavage reaction. In the presence of both ribozyme cleaving strands magnesium-ion dependent cleavage of a model 26 base beta APP substrate RNA or full-length beta APP-751 mRNA was observed at the hammerhead consensus cleavage site. Neither ribozyme was active against non-message homologs of beta APP mRNA, nor was cleavage detected when point mutations were made in the conserved core sequences. However, the kcat/Km at 37 degrees C in 10 mM Mg+2 of the longer ribozyme was reduced twenty-fold when model and full-length substrates were compared. The use of short deoxyoligonucleotides (13-17 mers) that bind upstream of the ribozyme was found to enhance the rate of cleavage of the full-length but not beta APP model substrate RNAs. The rate of enhancement depended on both the length of the deoxyoligonucleotide used as well as its site of binding with respect to the ribozyme. These data demonstrate the utility of ribozymes to cleave target RNAs in a catalytic, site-specific fashion in vitro. Direct comparison of the efficiency of different ribozyme constructs and different modulating activities provide an experimental strategy for designing more effective ribozymes for therapeutic purposes.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Denman</LastName><ForeName>R B</ForeName><Initials>RB</Initials><AffiliationInfo><Affiliation>New York Institute for Basic Research in Developmental Disabilities, Staten Island 10314.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>AGO4221</GrantID><Acronym>AG</Acronym><Agency>NIA NIH HHS</Agency><Country>United States</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType><PublicationType UI="D013487">Research Support, U.S. Gov't, 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