A combined in vitro / in vivo selection for polymerases with novel promoter specificities

BACKGROUND: The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter. A combined in vitro / in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities. Large (10(3) – 10(6)) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendent mRNAs in vivo. Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection. RESULTS AND CONCLUSIONS: A T7 RNA polymerase library that was randomized at three positions was cloned adjacent to a T3-like promoter sequence, and a 'specialist' T7 RNA polymerase was identified. A library that was randomized at a different set of positions was cloned adjacent to a promoter library in which four positions had been randomized, and 'generalist' polymerases that could utilize a variety of T7 promoters were identified, including at least one polymerase with an apparently novel promoter specificity. This method may have applications for evolving other polymerase variants with novel phenotypes, such as the ability to incorporate modified nucleotides

Modular control of multiple pathways using engineered orthogonal T7 polymerases

Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a ‘controller’ plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.United States. Office of Naval Research (Award number N00014-10-1-0245)National Science Foundation (U.S.). (CCF-0943385)National Institutes of Health (U.S.) (AI067699)National Science Foundation (U.S.). Graduate Research FellowshipAmerican Society for Engineering Education. National Defense Science and Engineering Graduate FellowshipHertz Foundation. Graduate Fellowshi

Aptamers as theranostic agents: modifications, serum stability and functionalisation

Aptamers, and the selection process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) used to generate them, were first described more than twenty years ago. Since then, there have been numerous modifications to the selectionprocedures. This review discusses the use of modified bases as a means of enhancing serum stability and producing effective therapeutic tools, as well as functionalising these nucleic acids to be used as potential diagnostic agents

Methylated Nucleobases: Synthesis and Evaluation for Base Pairing In Vitro and In Vivo

The synthesis, base pairing properties and in vitro (polymerase) and in vivo (E. coli) recognition of 2′‐deoxynucleotides with a 2‐amino‐6‐methyl‐8‐oxo‐7,8‐dihydro‐purine (X), a 2‐methyl‐6‐thiopurine (Y) and a 6‐methyl‐4‐pyrimidone (Z) base moiety are described. As demonstrated by Tm measurements, the X and Y bases fail to form a self‐complementary base pair. Despite this failure, enzymatic incorporation experiments show that selected DNA polymerases recognize the X nucleotide and incorporate this modified nucleotide versus X in the template. In vivo, X is mainly recognized as a A/G or C base; Y is recognized as a G or C base and Z is mostly recognized as T or C. Replacing functional groups in nucleobases normally involved in W−C recognition (6‐carbonyl and 2‐amino group of purine; 6‐carbonyl of pyrimidine) readily leads to orthogonality (absence of base pairing with natural bases)

Directed evolution of T7 RNA polymerase variants using an 'autogene'

Natural enzymes, when used in biotechnological applications are often found not well suited for the tasks. Enzyme properties can now be improved by rational design or by directed evolution to produce useful biocatalysts. The work described in this thesis is mainly focused towards developing a directed evolution method to evolve RNA polymerases with novel properties, such as the ability to use modified nucleotides as substrates. A variety of modified nucleotides can be imagined that can impart unique characteristics to RNA molecules into which they are incorporated. Modified nucleotides improve the functionality of RNA, but more importantly, they increase the stability of RNA towards nucleases thus lending the RNA amenable to various biotechnological applications. Modified RNA transcripts will also find applications in the in vitro selection of aptamers and ribozymes. We have developed a directed evolution method for the isolation of RNA polymerase variants with altered promoter specificities and novel substrate specificities vii using a construct called an “autogene”. The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNA polymerase) is being used for the directed evolution studies. In short, a library of T7 RNA polymerase variants was made by randomizing the gene of T7 RNA polymerase at amino acid positions that are important for a desired activity (for e.g.: altered promoter recognition). This gene was then cloned downstream of a T7 promoter, generating a so-called autogene library. Following transformation to E.coli, those polymerase variants that best recognized their adjacent promoter self-amplified both their mRNAs and themselves in vivo. The variant mRNAs extracted from the population as a whole will be roughly represented according to the activities of their corresponding variant polymerases. Following reverse transcription and PCR amplification in vitro, the most abundant polymerase genes were carried into subsequent rounds of selection. The method allows large (103 -106 ) polymerase libraries to be efficiently searched for their promoter recognition ability and fidelity. Autogene selection was subsequently modified with a reporter gene and used to screen polymerase variants that can incorporate modified nucleotides into the RNA backbone. We have successfully evolved a novel T7 RNA polymerase variant that transcribes 2’-O-methyl RNA. Other selections can also be envisioned using the autogene system to discover new polymerases with novel abilities. The polymerases thus evolved were used to construct modified RNA libraries to be used in in vitro selection of modified ribozymes.Chemistr

Polymerase Amplification, Cloning, and Gene Expression of Benzo‐Homologous “yDNA” Base Pairs

A widened DNA base pair architecture is studied in an effort to explore the possibility of whether new genetic system designs might possess some of the functions of natural DNA. In the “yDNA” system, pairs are homologated by addition of a benzene ring, yielding (in the present study), benzopyrimidines that are correctly paired with purines. Here we report initial tests of ability of the benzopyrimidines yT and yC to store and transfer biochemical and biological information in vitro and in bacterial cells. In vitro primer extension studies with two polymerases showed that the enzymes could insert the correct nucleotides opposite these yDNA bases, but with low selectivity. PCR amplifications with a thermostable polymerase resulted in correct pairings in 15–20% of the cases, and more successfully when yT or yC were situated within the primers. Segments of DNA containing one or two yDNA bases were then ligated into a plasmid and tested for their ability to successfully lead the expression of an active protein in vivo. Although active at only a fraction of the activity of fully natural DNA, the unnatural bases encoded the correct codon bases in the majority of cases when singly substituted, yielding functioning green fluorescent protein. Although the activities with native polymerases are modest with these large base pairs, this is the first example of encoding protein in vivo by an unnatural DNA base pair architecture