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.

 

Tool Developer Website Summary
mfold University of Albany http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form RNA folding software; folding temperature and ionic conditions are fixed
NUPACK California Institute of Technology http://www.nupack.org/ RNA software suite for design and folding analysis with the option of designing RNA reaction pathways
RNA Designer University of British Columbia http://www.rnasoft.ca/cgi-bin/RNAsoft/RNAdesigner/rnadesign.pl RNA design tool using the dot-bracket format; temperature and GC content are adjustable
RBS Calculator Penn State University https://salis.psu.edu/software/ Predicts translation initiation rate in bacteria; takes into account RNA secondary structures for predictions
Nucleotide BLAST National Center for Biotechnology Information http://blast.ncbi.nlm.nih.gov/Blast.cgi BLAST compares nucleotide sequences to sequence database and calculates the statistical significance of any match
Primer-BLAST National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/tools/primer-blast/ Uses the popular primer3 engine to design primers; results are submitted to BLAST to check for unwanted endogenous match
BioNumbers Harvard Medical School http://bionumbers.hms.harvard.edu/ Registry of useful biological numbers, including genomic GC contents
genormPLUS Biogazelle http://www.biogazelle.com/genormplus/ Algorithm to determine the most stable reference genes from a set of tested candidate reference genes in a given qPCR sample panel

JBEI-ICE, part registry platform and tools

The Joint BioEnergy Institute Inventory of Composable Elements (JBEI-ICEs) is an open source registry platform for managing information about biological parts. It is capable of recording information about ‘legacy’ parts, such as plasmids, microbial host strains and Arabidopsisseeds, as well as DNA parts in various assembly standards. ICE is built on the idea of a web of registries and thus provides strong support for distributed interconnected use. The information deposited in an ICE installation instance is accessible both via a web browser and through the web application programming interfaces, which allows automated access to parts via third-party programs. JBEI-ICE includes several useful web browser-based graphical applications for sequence annotation, manipulation and analysis that are also open source. As with open source software, users are encouraged to install, use and customize JBEI-ICE and its components for their particular purposes. As a web application programming interface, ICE provides well-developed parts storage functionality for other synthetic biology software projects. A public instance is available at public-registry.jbei.org, where users can try out features, upload parts or simply use it for their projects. The ICE software suite is available via Google Code (http://code.google.com/p/gd-ice/) , a hosting site for community-driven open source projects.

 

FX: an RNA-Seq analysis tool on the cloud

FX is an RNA-Seq analysis tool, which runs in parallel on cloud computing infrastructure, for the estimation of gene expression levels and genomic variant calling. In the mapping of short RNA-Seq reads, FX uses a transcriptome-based reference primarily, generated from ∼160,000 mRNA sequences from RefSeq, UCSC and Ensembl databases. This approach reduces the misalignment of reads originating from splicing junctions. Unmapped reads not aligned on known transcripts are then mapped on the human genome reference. FX allows analysis of RNA-Seq data on cloud computing infrastructures, supporting access through a user-friendly web interface.

FX is freely available on the web at (http://fx.gmi.ac.kr), and can be installed on local Hadoop clusters. Guidance for the installation and operation of FX can be found under the ‘Documentation’ menu on the website.