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

Computer Program Enrich Understandings to RNA motifs

DNA carries and passes the genetic information with nucleotides sequences, but in some virus RNA does the job instead of DNA.  In addition, the 3D structure of RNA, RNA motif, shows more various roles in cellular functions. It means that only with sequence information we cannot fully understand the RNA roles and functions.

Detecting and exploring RNA motif are prone to lie in modeling, engineering, and this is partly why we need computational biology.

RNA structural motifs are the building blocks of the complex RNA architecture. Identification of non-coding RNA structural motifs is a critical step towards understanding of their structures and functionalities. In this article, we present a clustering approach for de novo RNA structural motif identification. We applied our approach on a data set containing 5S, 16S and 23S rRNAs and rediscovered many known motifs including GNRA tetraloop, kink-turn, C-loop, sarcin–ricin, reverse kink-turn, hook-turn, E-loop and tandem-sheared motifs, with higher accuracy than the state-of-the-art clustering method. We also identified a number of potential novel instances of GNRA tetraloop, kink-turn, sarcin–ricin and tandem-sheared motifs. More importantly, several novel structural motif families have been revealed by our clustering analysis. We identified a highly asymmetric bulge loop motif that resembles the rope sling. We also found an internal loop motif that can significantly increase the twist of the helix. Finally, we discovered a subfamily of hexaloop motif, which has significantly different geometry comparing to the currently known hexaloop motif. Our discoveries presented in this article have largely increased current knowledge of RNA structural motifs.

Researchers at University of Central Florida [1] used a complex computer program to analyze RNA motifs — the subunits that make up RNA (ribonucleic acid).

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[1] C. Zhong, S. Zhang. Clustering RNA structural motifs in ribosomal RNAs using secondary structural alignment.Nucleic Acids Research, 2011; 40 (3): 1307 DOI:10.1093/nar/gkr804