System and process for in vivo manufacturing nanostructure-ended double-stranded covalently-closed linear DNA, the resulting molecules and their uses
AssigneesMartin Williams, Syste.Bio Inc.
InventorMartin Williams
Life of this patent
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Abstract
The present invention relates to a novel biological in vivo manufacturing system and a process for generating nanostructure-ended double-stranded covalently-closed linear DNA molecules. These DNA molecules possess the ability to merge the information-storage and function-encoding attributes of nucleic acids with the structural properties and functional capabilities typically found in proteins, such as specific binding and catalysis, in a single nucleic acid-only molecular entity, making them useful for a wide variety of medical applications and industrial implementations.
Claims7
A nanostructure-ended double-stranded covalently-closed linear DNA molecule in vivo manufacturing system comprising:
- a recombinant cell comprising:
- a repressor protein module comprising:
- a first bacterial constitutive promoter, a first ribosome binding site, a first repressor protein coding sequence, a second ribosome binding site, a second repressor protein coding sequence, and a first bacterial terminator;
- a recombinase-reverse transcriptase module comprising:
- a first bacterial inducible promoter, a third ribosome binding site, a recombinase coding sequence, a fourth ribosome binding site, a reverse transcriptase coding sequence, and a second bacterial terminator;
- and
- a homing endonuclease module comprising:
- a second bacterial inducible promoter, a fifth ribosome binding site, a homing endonuclease coding sequence, and a third bacterial terminator;
- and
- a parental plasmid DNA platform, housed in the recombinant cell, comprising:
- a retron module comprising:
- a left retron unit comprising:
- a first parental bacterial constitutive promoter, a multi-copy single-stranded RNA (msr) sequence, a 5′-multi-copy single-stranded DNA (msd) sequence, a left sense stem recombination sequence (L-SSR), a left DNA nanostructure sequence (L-DNS) inserted in a first multiple cloning site (MCS 1), a left antisense stem recombination sequence (L-ASR), complementary to the L-SSR sequence, a 3′-msd sequence, and a first parental bacterial terminator;
- and
- a right retron unit comprising:
- a second parental bacterial €constitutive promoter, a msr sequence, a 5′-msd sequence, a right sense stem recombination sequence (R-SSR), a right DNA nanostructure sequence (R-DNS) inserted in a second multiple cloning site (MCS 2), a right antisense stem recombination sequence (R-ASR), complementary to the R-SSR sequence, a 3′-msd sequence, and a second parental bacterial terminator;
- a linear module comprising:
- a first acceptor recombinase recognition sequence (A1), a sequence selected from a DNA spacer and an expression cassette inserted in a third multiple cloning site (MCS 3), and a second acceptor recombinase recognition sequence (A2);
- and
- a bacterial backbone comprising:
- a parental bacterial origin of replication, a parental selection marker, and a parental homing endonuclease recognition sequence (HRE),
- wherein the retron units, linear module, and bacterial backbone are contained in at least one cloned parental plasmid DNA.
Dependencies are inferred from the claim text.