Improved processes for in vitro transcription of messenger rna
AssigneeTranslate Bio, Inc.
InventorsDustin Cooper, Frank DeRosa, Anusha Dias, Jeffrey S. Dubins, Xiaobo Gu, Khang Anh Tran
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Abstract
The present invention provides methods for preparing optimized DNA sequences as templates for in vitro transcription of mRNA. These DNA sequences are optimized to avoid premature termination of transcription by RNA polymerase. The invention also provides methods for preparing optimized DNA sequences that include one or more termination signal at their 3′ end to reduce or prevent non-templated “runoff” transcription.
Claims36
A method for preparing an optimized DNA sequence encoding a protein as a template for in vitro transcription, said method comprising:
- a. providing a DNA sequence that comprises a protein coding sequence;
- b. determining the presence of a termination signal in the DNA sequence, wherein the termination signal has the following nucleic acid sequence:
- 5′-X1ATCTX2TX3-3′, wherein X1, X2 and X3 are independently selected from A, C, T or G;
- and
- c. if one or more termination signal is present, modifying the DNA sequence by replacing one or more nucleic acids at any one of position 2, 3, 4, 5 and 7 of said termination signal(s) with any one of the other three nucleic acids to generate the optimized DNA sequence, wherein, if required, the one or more replacement nucleic acids are selected to preserve the amino acid sequence of the protein encoded by the protein coding sequence.
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A method for preparing an optimized DNA sequence encoding a protein as a template for in vitro transcription, said method comprising:
- a. providing a DNA sequence encoding a protein;
- and
- b. adding one or more termination signals at the 3′ end of the DNA sequence to provide the optimized DNA sequence,
- wherein the one or more termination signal(s) comprises the following nucleic acid sequence:
- 5′-X1ATCTX2TX3-3′, wherein X1, X2 and X3 are independently selected from A, C, T or G.
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A DNA sequence for use in in vitro transcription, comprising in 5′ to 3′ order:
- a. A 5′UTR;
- b. a protein coding sequence;
- c. a 3′UTR;
- d. optionally a nucleic acid sequence encoding a polyA tail;
- and
- e. a termination signal;
- wherein the termination signal comprises the following nucleic acid sequence:
- 5′-X1ATCTX2TX3-3′, wherein X1, X2 and X3 are independently selected from A, C, T or G.
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Dependencies are inferred from the claim text.