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 … Read moreDesigning and using RNA scaffolds to assemble proteins in vivo

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 … Read moreA Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

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 … Read moremicroRNAs quantitative assay with Splinted Ligation