Transfection kinetics and structural promoters
AssigneeNucleonics, Inc.
InventorsCatherine J. Pachuk, Chandrasekhar Satishchandran
Life of this patent
Estimating the term…
Abstract
The invention features methods of analyzing the kinetics properties of transfection reactions. Also featured are methods for creating structural promoters which are effectively unregulated by enhancers and repressors. The structural promoters are significantly more active than the native promoter sequences upon which they are based.
Claims121
A method for producing a forced-open promoter complex, wherein said forced-open promoter complex promotes an increase in transcription of a nucleic acid sequence of interest, said method comprising:
- (i) providing a circular supercoiled DNA comprising the sequence of interest,
- (ii) annealing to said DNA an oligonucleotide having a 5′ end, a 3′ end, and region linking the 5′ and 3′ ends, the 5′ end being complementary to a first region of the DNA that is upstream of said sequence of interest relative to the direction of transcription, and the 3′ end being complementary to a second region of the DNA that is contiguous to said first region;
- such that the 5′ and 3′ ends are annealed to adjacent bases of the DNA;
- and
- (iii) circularizing the 5′ end of said oligonucleotide to the 3′ end of said oligonucleotide to topologically link the oligonucleotide to the DNA, thereby producing a forced-open promoter complex and promoting transcription of said sequence of interest.
A method for increasing the transcription of a nucleic acid sequence comprising:
- (i) forming a forced open promoter complex in a supercoiled DNA comprising said nucleic acid sequence;
- (ii) transfecting the resulting supercoiled DNA comprising said forced open promoter complex into a cell;
- and
- (iii) culturing said cell, wherein said forced open promoter complex promotes increased transcription of said nucleic acid sequence, relative to the transcription of a nucleic acid sequence in a cell transfected with a supercoiled DNA lacking a forced open promoter complex.
A composition comprising:
- a circular supercoiled DNA containing a sequence for expression;
- and
- a padlocked oligonucleotide annealed upstream of said sequence for expression relative to the direction of transcription, so as to enhance transcription of said sequence for expression relative to the transcription of said sequence from a supercoiled DNA lacking a padlocked oligonucleotide.
A composition comprising an HCMV promoter annealed to a torsionally locked oligonucleotide.
A chimeric HCMV/EF-1α Pmin promoter.
A composition comprising a supercoiled DNA comprising a promoter sequence annealed to a torsionally locked oligonucleotide, wherein said promoter sequence is operably linked to a nucleic acid sequence.
A method for generating an open-promoter complex, comprising:
- (i) providing a purified plasmid in supercoiled form, the plasmid containing a sequence for expression;
- (ii) creating a padlocked oligonucleotide to generate an open-promoter complex for said sequence for expression.
A composition, comprising:
- a purified plasmid DNA in supercoiled form, the construct containing a sequence for expression;
- and
- a padlocked oligonucleotide annealed to said construct, wherein the padlocked oligonucleotide generates an open-promoter complex for said sequence for expression.
Dependencies are inferred from the claim text.