Dynamic control of colocalization of proteins

#The Sequence Listing for this application is labeled “UOD-512US_SequenceUsting.txt” which was created on Feb. 24, 2020 and is 841 Bytes. The entire content of the sequence listing is incorporated herein by reference in its entirety.

#The invention relates to colocalization of proteins in cells via scaffold RNA molecules having complementary sequences, especially proteins capable of providing a biological activity when colocalized or not colocalized depending on the nature of the interaction between the proteins.

#Through metabolic engineering, microorganisms have been engineered to produce high-value chemicals ranging from biofuels to pharmaceuticals. Traditionally, production of non-native molecules has been achieved by a two-step process: (a) introduce a heterologous metabolic pathway into a model organism, such as E. coli and S. cerevisiae, and (b) control the metabolic flux through the non-native pathway through genetic insertions, substitutions, and deletions to regulate enzyme expression. Simple introduction of the enzymes in a metabolic pathway of interest through genetic vectors has been successful for molecules such as small chain hydrocarbons. However, tedious and often unrewarding optimization of the turnover and expression rates of the introduced enzyme is often required to minimize the stress the new pathway inflicts on the model organism.

#FIG. 1 is an illustration of the concept of substrate channeling. When enzymes E1, E2, and E3 are in close proximity, i.e., colocalized, the flux through the metabolic pathway as a whole increases significantly compared to when they are not colocalized.

#FIG. 2 illustrates one embodiment of toehold mediated strand displacement (TMSD) according to the invention in four drawings from left to right. The first drawing shows a complex, in which a top strand comprises Sequence 1 and a bottom strand comprises Sequence 1* and Sequence 2* at the 3′ end of Sequence 1*, and a trigger strand comprising Sequence 2 and Sequence 1 at the 3′ end of Sequence 2. Sequence 2* is a toehold region. Sequence 1* in the bottom strand is complementary with and hybridizes to Sequence 1 in the top strand. Sequence 2* is complementary to Sequence 2. The second drawing shows initiation of TMSD by hybridization of Sequence 2 in the trigger strand to Sequence 2* in the bottom strand. The third drawing shows that TMSD continues with hybridization of Sequence 1 in the Trigger Sequence to Sequence 1* in the bottom strand. The fourth drawing shows complete TMSD of the top strand by the trigger strand. The arrows represent the 3′ end of each strand.

#FIG. 3 illustrates a variety of Cas proteins. (a) Type 1 CRISPR/Cas systems. Cas 6 processes long RNA to small crRNA and triggers complex formation. (b) Type II CRISPR/Cas systems. Hybridization of tracrRNA and crRNA to each other allows Cas9 to bind to the complex and identify target DNA sequences. (c) Type III CRISPR/Cas system. Cas5 processes RNA to crRNA and triggers complex formation. Cas6 protein from the CRISPR/Cas Type I family is one of the smaller proteins and exemplified in examples 1, 2, 6 and 7.

#The present invention relates to methods for controlling colocalization of two or more proteins via RNA scaffold systems. The invention is especially useful for proteins that interact with each other or are involved in the same biological pathway in the fields of, for example, metabolic engineering and synthetic biology. Genetic circuitry developed using methods of the present invention from synthetic biology helps control the metabolic flux in cells in order to maximize titer product and maximize cell viability. The scaffolds developed according to this invention may be used to colocalize various proteins, for example, ranging from reporter systems to nonnative metabolic pathway proteins, and improve titers in a variety of small molecule chemicals.

#The inventors have surprisingly discovered methods for assemble or disassemble a scaffold for controlling colocalization of two Cas6 fusion proteins through hybridization by two RNA strands having complementary sequences and bound specifically to the two Cas6 and/or through toehold mediated strand displacement (TMSD). Proteins having high binding affinities to specific RNA sequences and de novo designed scaffold RNA molecules comprising such specific RNA sequences have been prepared for controlling colocalization of the proteins in vivo and providing biological activity due to colocalization of the proteins. The scaffold may dynamically assemble and bring the fusion proteins of interest, in proximity, for example, within 5-10 nm. Once the scaffold has assembled, the disassembly may be triggered through TMSD, a technology that allows for displacing one DNA or RNA strand in favor of a new trigger strand. The displacement may be facilitated by the presence of an unhybridized 6-12 nucleotide long toehold region at the end of one of the initially hybridized strands. The toehold region provides a foothold onto which the trigger strand can begin to hybridize on. Eventually, the trigger strand may completely hybridize with the toehold strand, essentially kicking out the second strand. The mechanism of TMSD is illustrated in FIG. 2.

#TMSD is an excellent candidate for facilitating the dynamic disassembly of the scaffold because its kinetics occurs in the order of minutes to hours. Furthermore, the kinetics can be fine-tuned by altering the length of the toehold and/or by introducing mismatched base pairs in the toehold region. Overall, the scaffold components, which are proteins bound to RNA molecules, may dynamically assemble through strand complementarity of the RNA molecules and, then, dynamically disassemble by the addition/production of a trigger strand. Although initially the trigger strand may most likely be synthetic, in the long run it is desirable that the trigger strand is under control of a native cell response (for example, low glucose levels or a stress response) so that dissociation of the scaffold may be controlled by cellular responses.