Stabilization of poly(A) sequence encoding DNA sequences
The present invention relates to nucleic acid molecules containing poly (dA:dT) regions which are stabilized in E. coli , methods of propagating such nucleic acid molecules in E. coli , methods of obtaining RNA, peptides or proteins using such nucleic acid molecules and to RNA which is obtained from such nucleic acid molecules and its use. In particular, the poly (dA:dT) regions contain at least one disruption by a sequence not encoding a sequence solely composed of A residues.
1. A nucleic acid molecule comprising in the 5′→3′ direction of transcription:
(a) a promoter;
(b) a transcribable nucleic acid sequence or a nucleic acid sequence for introducing a transcribable nucleic acid sequence; and
(c) a nucleic acid sequence which, when transcribed under the control of the promoter (a), codes for a modified polyadenyl sequence of at least 80 consecutive nucleotides, wherein the modified polyadenyl sequence comprises:
a linker sequence comprising at least one T, C, or G nucleotide;
a first sequence of at least 20 A consecutive nucleotides, which is 5′ of the linker sequence; and
a second sequence of at least 20 A consecutive nucleotides, which is 3′ of the linker sequence.
2. The nucleic acid molecule as claimed in claim 1 , wherein the linker sequence is a sequence of 2 or more consecutive nucleotides, wherein the first and the last nucleotide of said sequence of 2 or more consecutive nucleotides is a nucleotide selected from the group consisting of T, C, and G.
3. The nucleic acid molecule as claimed in claim 1 , wherein the modified polyadenyl sequence of at least 80 consecutive nucleotides comprises at least 90 nucleotides.
4. The nucleic acid molecule as claimed in claim 1 , wherein the linker sequence is located within a region from position 21 to position 80 of said modified polyadenyl sequence.
5. The nucleic acid molecule as claimed in claim 2 , wherein the linker sequence has a length of at least 3 nucleotides.
6. The nucleic acid molecule as claimed in claim 2 , wherein said sequence of 2 or more consecutive nucleotides comprises 3 or fewer consecutive A nucleotides.
7. The nucleic acid molecule as claimed in claim 1 , wherein the nucleic acid sequences (b) and (c) under the control of the promoter (a) can be transcribed to give a common transcript.
8. The nucleic acid molecule as claimed in claim 1 , wherein the modified polyadenyl sequence of at least 80 consecutive nucleotides is located at the 3′ end of a transcript.
9. The nucleic acid molecule as claimed in claim 1 , which is a closed circular molecule or a linear molecule.
10. The nucleic acid molecule as claimed in claim 1 , comprising the transcribable nucleic acid sequence, which comprises a nucleic acid sequence coding for a peptide or protein.
11. The nucleic acid molecule as claimed in claim 1 , further comprising one or more members selected from the group consisting of: (i) a reporter gene; (ii) a selectable marker; and (iii) an origin of replication.
12. The nucleic acid molecule as claimed in claim 1 , which is suitable, after linearization, for in vitro transcription of mRNA.
13. A method of propagating a nucleic acid molecule, comprising:
(i) providing a nucleic acid molecule as claimed in claim 1 , and
(ii) propagating said nucleic acid molecule in Escherichia coli.
14. The method of claim 13 , wherein propagating said nucleic acid molecule in Escherichia coli comprises transforming Escherichia coli with said nucleic acid molecule and cultivating said transformed Escherichia coli.
15. The method of claim 13 , which further comprises isolating said nucleic acid molecule from Escherichia coli following propagation.
16. A method of obtaining RNA, comprising:
(i) propagating a nucleic acid molecule according to the method of claim 13 , and
(ii) transcribing in vitro the nucleic acid molecule as a template into RNA.
17. A method of obtaining a peptide or protein, comprising:
(i) obtaining mRNA encoding a peptide or protein according to the method of claim 16 , and
(ii) translating the mRNA into the peptide or protein sequence.
18. The method as claimed in claim 16 , further comprising, prior to (ii), cleaving the nucleic acid molecule.
19. The nucleic acid molecule as claimed in claim 1 , comprising the nucleic acid sequence for introducing a transcribable nucleic acid sequence, which comprises a multiple cloning site.