Ribozymes (ribonucleic acid enzymes) are RNA molecules that are capable of catalyzing specific biochemical reactions, similar to the action of protein enzymes. The most common activities of natural or in vitro-evolved ribozymes are the cleavage or ligation of RNA and DNA and peptide bond formation. They also participate in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. The figure to the left depicts a hairpin ribozyme.
We have leveraged this incredible technology to create a unique ribozyme design that is capable of discovering all active promoters in an organisms’ genome so that they can be analyzed and then evolved for enhanced performance. Our unique design includes a very small plasmid that contains a ribozyme core. The ribozyme core cleaves transcriptionally active nucleotide sequences and ligates those sequences back into the small plasmid. Furthermore, the ribozyme forces the promoter sequence in the plasmid to circularize, protecting itself from nuclease activity. Our method requires no foreknowledge of an organism’s transcription control sequences or coding sequences of any specific genes. It can be applied to the genomic DNA of organisms from any kingdom, and does not require a reporter gene detection scheme. It can also be applied to entirely synthetic sequences to form artificial transcription control sequences. We also have the ability to evolve any transcription control sequence that we discover, and these evolved sequences are not bound by the US Supreme Court’s Myriad decision, and as such, can be patented.
HOW IT WORKS: PROMOTER DISCOVERY
A promoter is a region of DNA that initiates transcription of a gene. Proximal promoters are located upstream, and are typically within 1Kb of the transcription start site. Distal promoters can contain additional regulatory elements, and can even be located on separate chromosomes. The biggest challenge with promoter discovery using current methods is that researchers don’t know where the promoter sequences are located. This forces trial-and-error, shot-in-the-dark methods of discovery that result in expensive, lengthy, and inefficient processes.
Our discovery process starts with genomic DNA from the species of interest. Naturally occurring promoter sequences are in that genomic DNA, we just don’t know where they are yet. We break the genomic DNA into discrete fragments and clone those fragments into our plasmid with the ribozyme core. The result is a library of genomic DNA elements that are transformed into the host species. Once inside cells, the ribozyme takes advantage of the hosts’ transcriptional machinery and produces copies with promoter sequences, where the total copy numbers of promoter sequences are proportional to the strength of the promoter. Think of each promoter sequence as an individual experiment, and all the promoter experiments are performed in parallel in a single population of cells. After an appropriate incubation time, the cells are lysed, and plasmids are prepped and sequenced. At the end of our discovery process, we have captured, sequenced, and measured the relative strength of every promoter that was active in the cell population. Because the transformation and recovery steps are handled in single reaction steps for all promoters, our method is exceptionally efficient and only takes 3-4 weeks.
HOW IT WORKS: PROMOTER ENHANCEMENT
Using a known promoter, we mutagenize the sequence to generate millions of unique promoter sequences and create a pool of mutant promoters that are ligated into our ribozyme plasmid to create a mutant library. The mutant library is transformed back into the host species in the same way we perform the discovery process. Each plasmid in the mutant library will now interact with the transcriptional machinery of the host cell, and our final sequencing readout tells us the sequence and relative strength of all the mutant promoters present in the host species.
The enhanced promoter sequencing readout tells us which promoter sequences will produce the greatest amount of transcript for a particular gene. To test the protein producing capacity of the promoters, the promoter sequences are cloned upstream of a gene of interest using an expression plasmid specific to the species of interest. We then compare expression levels to the wild type promoter in the same expression plasmid.
For protein validation in bacteria, we use maltose binding protein (MBP) in a pUC plasmid transformed into an E. coli host. Because the enhanced promoters drive expression between 1-1000% of the wild type promoter, we measure the expressed proteins using an ELISA assay to ensure accurate measurement of each enhanced promoter.
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