Designing and using RNA scaffolds to assemble proteins in vivo

RNA scaffolds are synthetic noncoding RNA molecules with engineered 3D folding harnessed to spatially organize proteins in vivo. Here we provide a protocol to design, express and characterize RNA scaffolds and their cognate proteins within 1 month. The RNA scaffold designs described here are based on either monomeric or multimeric units harboring RNA aptamers as protein docking sites. The scaffolds and proteins are cloned into inducible plasmids and expressed to form functional assemblies. RNA scaffolds find applications in many fields in which in vivo organization of biomolecules is of interest. RNA scaffolds provide extended flexibility compared with DNA or protein scaffolding strategies through programmed modulation of multiple protein stoichiometry and numbers, as well as the proteins’ relative distances and spatial orientations. For synthetic biology, RNA scaffolds provide a new platform that can be used to increase yields of sequential metabolic pathways.


mfoldUniversity of Albany folding software; folding temperature and ionic conditions are fixed
NUPACKCalifornia Institute of Technology software suite for design and folding analysis with the option of designing RNA reaction pathways
RNA DesignerUniversity of British Columbia design tool using the dot-bracket format; temperature and GC content are adjustable
RBS CalculatorPenn State University translation initiation rate in bacteria; takes into account RNA secondary structures for predictions
Nucleotide BLASTNational Center for Biotechnology Information compares nucleotide sequences to sequence database and calculates the statistical significance of any match
Primer-BLASTNational Center for Biotechnology Information the popular primer3 engine to design primers; results are submitted to BLAST to check for unwanted endogenous match
BioNumbersHarvard Medical School of useful biological numbers, including genomic GC contents
genormPLUSBiogazelle to determine the most stable reference genes from a set of tested candidate reference genes in a given qPCR sample panel

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