Modified promoter for RNA polymerase, its preparation and its applications
First Claim
1. An oligonucleotide that serves as a promoter non-template strand in transcription of a sequence of a nucleotide target, in the presence of a phage RNA polymerase, from a site of the target which is not normally a transcription start site for the phage RNA polymerase, said polymerase having a specific natural promoter comprising a consensus sequence at least from position -17 to position -1, wherein:
- said oligonucleotide comprises a core sequence flanked on at least one of its ends by a nucleotide sequence capable of hybridization with a sequence of the target,said core sequence consists of a sequence of 6 to 9 consecutive nucleotides chosen from the region -12 to -4 of the non-template strand of the specific natural promoter, or a sufficiently homologous sequence to enable the functionality of the RNA polymerase to be retained,at least a first flanking sequence is complementary to a first region of the target, and a second flanking sequence, when present, is complementary to a second region of the target, said first and second regions being separated on the target by a sequence having a number of nucleotides equal to the number of nucleotides in the core sequence, andthe number of nucleotides in the flanking sequence, or the sum of the number of nucleotides in the flanking sequences, is at least sufficiently high to enable the oligonucleotide to hybridize with the target at a temperature of use of the RNA polymerase.
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Abstract
An oligonucleotide is intended to be used as a promoter non-template strand in the transcription of a sequence of a nucleotide target in the presence of a phage RNA polymerase. The phage RNA polymerase has specific natural promoters containing a consensus sequence from at least position -17 to position -1. The oligonucleotide contains a core sequence flanked at at least one of its ends by a nucleotide sequence capable of hybridization with a sequence of the target. The core sequence contains a sequence of 6 to 9 consecutive nucleotides from the region -12 to -4 of the non-template strand of the specific promoter, or a sufficiently homologous sequence to enable the functionality of the RNA polymerase to be retained. One flanking sequence is complementary to a first region of the target, and a second flanking sequence, when present, is complementary to a second region of the target, the first and second regions being separated on the target by a sequence having a number of nucleotides equal to the number of nucleotides in the core sequence. The number of nucleotides in the flanking region, or the sum of the number of nucleotides in the flanking regions, is at least sufficiently high for the nucleotide to be able to hybridize with the target at the temperature of use of the RNA polymerase. Such an oligonucleotide enables transcription to be initiated at a site of the target which is not normally a transcription start site for the RNA polymerase.
30 Citations
16 Claims
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1. An oligonucleotide that serves as a promoter non-template strand in transcription of a sequence of a nucleotide target, in the presence of a phage RNA polymerase, from a site of the target which is not normally a transcription start site for the phage RNA polymerase, said polymerase having a specific natural promoter comprising a consensus sequence at least from position -17 to position -1, wherein:
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said oligonucleotide comprises a core sequence flanked on at least one of its ends by a nucleotide sequence capable of hybridization with a sequence of the target, said core sequence consists of a sequence of 6 to 9 consecutive nucleotides chosen from the region -12 to -4 of the non-template strand of the specific natural promoter, or a sufficiently homologous sequence to enable the functionality of the RNA polymerase to be retained, at least a first flanking sequence is complementary to a first region of the target, and a second flanking sequence, when present, is complementary to a second region of the target, said first and second regions being separated on the target by a sequence having a number of nucleotides equal to the number of nucleotides in the core sequence, and the number of nucleotides in the flanking sequence, or the sum of the number of nucleotides in the flanking sequences, is at least sufficiently high to enable the oligonucleotide to hybridize with the target at a temperature of use of the RNA polymerase. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for preparing oligonucleotides that serve as promoter non-template strands in transcription of a sequence of a nucleotide target, in the presence of a phase RNA polymerase, from a site which is not normally a transcription start site for the phage RNA polymerase, said polymerase having a specific natural promoter comprising a consensus sequence at least from position -17 to position -1, comprising:
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(1) synthesizing oligonucleotides comprising a core sequence flanked on at least one of its ends by a nucleotide sequence capable of hybridization with a sequence of the target, wherein said core sequence consists of a sequence of 6 to 9 consecutive nucleotides chosen from the region -12 to -4 of the non-template strand of the specific natural promoter, or a sufficiently homologous sequence to enable the functionality of the RNA polymerase to be retained, at least a first flanking sequence is complementary to a first region of the target, and a second flanking sequence, when present, is complementary to a second region of the target, said first and second regions being separated on the target by a sequence having a number of nucleotides equal to the number of nucleotides in the core sequence, and the number of nucleotides in the flanking sequence, or the sum of the number of nucleotides in the flanking sequences, is at least sufficiently high to enable the oligonucleotide to hybridize with the target at a temperature of use of the RNA polymerase, (2) performing tests of transcription of the target using the oligonucleotides obtained, and (3) eliminating oligonucleotides which do not enable transcription to be effected with a yield at least equal to a predetermined threshold, thereby preparing said oligonucleotides that serve as promoter non-template strands in transcription of a sequence of a nucleotide target, in the presence of a phase RNA polymerase, from a site which is not normally a transcription start site for the phage RNA polymerase. - View Dependent Claims (14, 15, 16)
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Specification
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Current AssigneeBio Merieux
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Original AssigneeBio Merieux
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InventorsCleuziat, Philippe, Mallet, Francois, Levasseur, Pierre, Guillou-Bonnici, Francoise
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Primary Examiner(s)JONES, WILLIAM G
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Assistant Examiner(s)Tran, Paul B.
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Application NumberUS08/360,051Time in Patent Office1,568 DaysField of Search435/6, 435/91.1, 435/91.2, 435/91.21, 435/91.5, 536/24.1, 536/24.33, 935/77, 935/78US Class Current435/91.1CPC Class CodesC12N 15/11 DNA or RNA fragments; Modif...C12P 19/34 Polynucleotides, e.g. nucle...C12Q 1/6865 Promoter-based amplificatio...C12Q 1/703 Viruses associated with AIDS
