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

A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

 

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http://www.sciencemag.org/content/337/6096/816.full

— later I’ll explain a little bit.

Maximum expected accuracy structural neighbors of an RNA secondary structure

Since RNA molecules regulate genes and control alternative splicing by allostery, it is important to develop algorithms to predict RNA conformational switches. Some tools, such as paRNAss, RNAshapes and RNAbor, can be used to predict potential conformational switches; nevertheless, no existent tool can detect general (i.e., not family specific) entire riboswitches (both aptamer and expression platform) with accuracy. Thus, the development of additional algorithms to detect conformational switches seems important, especially since the difference in free energy between the two metastable secondary structures may be as large as 15-20 kcal/mol. It has recently emerged that RNA secondary structure can be more accurately predicted by computing the maximum expected accuracy (MEA) structure, rather than the minimum free energy (MFE) structure.

 

Source code for RNAborMEA can be downloaded from http://sourceforge.net/projects/rnabormea/ or http://bioinformatics.bc.edu/clotelab/RNAborMEA/

microRNAs quantitative assay with Splinted Ligation

Do you still use Northern Blot to quantitate microRNAs expression? Here I recommend Splinted Ligation Assay[1,2], though this method is an old story (published in 2007).

 

Detection of miRNAs using splinted ligation. Schematic depiction of the assay process. As described in the text, the assay involves: (1) Labeling of the ligation oligonucleotide; (2) concurrent annealing of the ligation oligonucleotide and miRNA to a bridge oligonucleotide; (3) linking of the ligation oligonucleotide to the miRNA by DNA ligase; (4) removal of labeled phosphate from unligated oligonucleotide; and (5) fractionation on a denaturing gel.

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[1] Maroney, P. a, Chamnongpol, S., Souret, F., & Nilsen, T. W. (2007). A rapid, quantitative assay for direct detection of microRNAs and other small RNAs using splinted ligation. RNA (New York, N.Y.), 13(6), 930-6. doi:10.1261/rna.518107

[2] Maroney, P. A., Chamnongpol, S., Souret, F., & Nilsen, T. W. (2008). Direct detection of small RNAs using splinted ligation. Nat. Protocols, 3(2), 279-287. Nature Publishing Group. Retrieved from http://dx.doi.org/10.1038/nprot.2007.530