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 method of expressing a peptide or polypeptide in a cell comprising transfecting a cell with a composition, wherein the composition comprises an mRNA comprising in the 5′→3′ direction:
a 5′-untranslated region (UTR);
a sequence encoding a peptide or polypeptide, wherein the 5′-UTR is heterologous to the sequence encoding the peptide or polypeptide; and
a modified polyadenyl sequence of at least 80 consecutive nucleotides, wherein the modified polyadenyl sequence comprises:
a first sequence of at least 20 A consecutive nucleotides;
a linker sequence comprising at least one U, C, or G nucleotide; wherein the linker sequence is a sequence of two to ten consecutive nucleotides, wherein the first and the last nucleotide of said sequence of two to ten consecutive nucleotides is a nucleotide selected from the group consisting of U, C, and G; and
a second sequence of at least 20 A consecutive nucleotides
wherein the linker sequence is between the first sequence and the second sequence.
2. The method of claim 1 , wherein the modified polyadenyl sequence of at least 80 consecutive nucleotides comprises at least 90 nucleotides.
3. The method of claim 1 , wherein the linker sequence is located within a region from position 21 to position 80 of said modified polyadenyl sequence.
4. The method of claim 1 , wherein the linker sequence has a length of at least 3 nucleotides.
5. The method of claim 1 , wherein said linker comprises 3 or fewer consecutive A nucleotides.
6. The method of claim 1 , wherein the mRNA is an in-vitro transcribed mRNA.
7. The method of claim 1 , wherein the modified polyadenyl sequence of at least 80 consecutive nucleotides is located at the 3′ end of the mRNA.
8. The method of claim 1 , wherein the mRNA further comprises a sequence encoding a reporter gene and/or a selectable marker.
9. The method of claim 1 , wherein the cell is an antigen-presenting cell.
10. The method of claim 1 , wherein the antigen-presenting cell is a dendritic cell, a monocyte or a macrophage.
11. The method of claim 1 , wherein the peptide or polypeptide is or comprises an antigen.
12. The method of claim 11 , wherein the antigen is or comprises a disease-associated antigen.
13. The method of claim 12 , wherein the disease-associated antigen is or comprises a tumor-associated antigen.
14. The method of claim 12 , wherein the disease-associated antigen is or comprises a viral antigen.
15. The method of claim 12 , wherein the disease-associated antigen is or comprises a bacterial antigen.
16. The method of claim 1 , further comprising a pharmaceutically acceptable excipient.