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

Synthetic Biology in Mammalian cells

Last post I mentioned an interesting research introducing RNA interfere system in bacterium and archaea. It gives a new sight into how similarities the three kingdoms share, and potentially what have been done in mammalian cells can be applied into E.coli to enrich the toolbox of synthetic biologists.

Today let’s take a glimpse at what is on earth the progress in mammalian synthetic biology world. Is the bio-system as feasible to engineer as E.coli, just like iGEM? Is the field quite matured enough? Or still long way to go?

As usual, I chase a line and here share out the reviews that probably gives me the answer.

  1. Weber, W., & Fussenegger, M. (2009). Engineering of synthetic mammalian gene networks. Chemistry & biology, 16(3), 287-97. Elsevier Ltd. doi:10.1016/j.chembiol.2009.02.005
  2. Weber, W., & Fussenegger, M. (2010). Synthetic gene networks in mammalian cells. Current opinion in biotechnology, 21(5), 690-6. Elsevier Ltd. doi:10.1016/j.copbio.2010.07.006
  3. Greber, D., & Fussenegger, M. (2010). An engineered mammalian band-pass network. Nucleic acids research, 38(18), e174. doi:10.1093/nar/gkq671
  4. Weber, W., & Fussenegger, M. (2011). Molecular diversity–the toolbox for synthetic gene switches and networks. Current opinion in chemical biology, 15(3), 414-20. Elsevier Ltd. doi:10.1016/j.cbpa.2011.03.003
  5. Weber, W., & Fussenegger, M. (2011). Emerging biomedical applications of synthetic biology. Nature Reviews Genetics, 13(1), 21-35. Nature Publishing Group. doi:10.1038/nrg3094
  6. Karlsson, M., Weber, W., & Fussenegger, M. (2012). Design and construction of synthetic gene networks in mammalian cells. Methods in molecular biology (Clifton, N.J.), 813, 359-76. Humana Press. doi:10.1007/978-1-61779-412-4_22

Based on the above researches, things in mammalian cells are not splendid engineered as E.coli, probably due to our limited understanding towards eukaryotes.

Nevertheless, there still are some sparkling researches. Here I raise one for example — Rapid Eraser, or precisely, Auxin-controlled protein depletion device.

Though it’s an old story, the bio-eraser inspires a lot. Another real old story is bio-film, the noted first E.coli photograph. An awkward problem  is the E.coli bio-films are ONCE-only. If you need another photo, you need buy one more new film .  Any modification? Protein Depletion!

Furthermore, let me explain why protein depletion device is wonderful first. Since it’s easy to enable E.coli express different color with natural dye seen under naked eye (seen E.chromi), or with GFP/RFP under UV light, what about rainbow sparkling E.coli Biofilm? The biofilm is more like a neon light. The E.coli itself can change its color from red to yellow, to green, and back to red periodically.


It’s Rainbow E.coli !!!

So how the protein depletion device works? Degron !!! A degron is a specific sequence of amino acids in a protein that directs the starting place of degradation. Once activated by ubiquitylation, for example, the protein will be rapidly degraded, thus seems to be erased.

As for auxin, auxin is employed as the inducing signal. As auxin-triggered degron system is conserved in yeast, avian and mammalian cells, it can be applied to yeast cells, and will not interfere with other proteins as signal noise or lead to fatal error.

What ‘s the speed? 97% depletion in 15~30min ! Very satisfying.

It is recommended that you read the paper [1] for further details.


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[1] Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T., & Kanemaki, M. (2009). An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nature methods, 6(12), 917-22. Nature Publishing Group. doi:10.1038/nmeth.1401


“RISC” of Bacteria and Archaea

Restriction-modification systems, abortive-phage phenotypes, toxin-antitoxins and other innate defense systems, in the past, have been shown in familiar chapters in typical microbiology textbook, while now what if I say in prokaryotes world “RISC” can serve a role for new kind of antiviral defense, in addition the “RNAi” can even be engineered and designed to lead to target gene silencing, would you believe me?

You must have ever heard CRISPR/Cas (CRISPR Associated proteins) System if you have ever read this Science paper [1]. Exactly as the title said, CRISPR, Clustered Regularly Inter-spaced Short Palindromic Repeat, serves as the leading role to provide the “memory” as an adaptive immunity, akin to a blacklist of unwanted visitors, like plasmids or viruses genome.

CRISPR/Cas has different types based on Cas family. Three modules of Cas proteins are Cmr, Cst, Csa. It is an old story in bacteria world as it had been firstly identified in E.coli in 1987. Most have been reported to head for invading DNA, while here what I introduce to you now is an unique and intriguing discovery that in achaeon Pyrococcus furiosus which thrives best under extremely high temperatures, CRISPR/Cmr (one subtype of CRISPR/Cas) targets invading RNA, rather than DNA, thus what I called “RNAi” can makes sense.

In general, the context of CRISPR RNA (crRNA) is typically a sandwich, repeat-sequence-repeat. The internal sequence is termed guide sequence which is complementary to invading RNA only for CRISPR/Cmr, and it is identical to invading DNA for most other cases of CRISPR/Cas.

–How CRISPR/Cmr works?


As recalled, the internal sequence of crRNA is complementary to invading RNA as the “seed” region, and more importantly only Cmr complex can contribute to RNA cleavage.

The 8 nt 5′-end tag, among the short repeat sequence in crRNA, will lead crRNA to bind to invading RNA. It is suggested that the 8 nt 5′-end tag plays a discrimination function to classify self-RNA and non-self RNA.

Once crRNA and invading RNA get paired, hydrolysis of the target RNA takes place at a fixed distance, 14nt, from the 3′-end of the small guide RNA. Thus the invading RNA will be degraded and its expression will be turned OFF. In this way Pyrococcus furiosus help themselves against foreign viruses invading with RNA gnome.

Thus I cannot help raising an analogy between CRISPR/Cas system and noted RISC (RNA-induced siliencing complex) in eukaryotes [2]. They all have the progess: processing to be matured, base-pair induced target cleavage.

–Can CRISPR/Cmr engineered?

Yes, we can. The magic is the 8nt 5′-end tag, whose sequence is AUUGAAAG. Scientists had hacked the “immune” system to suppress target gene expression [3], here with the example beta-lactamase (bla) mRNA. The internal sequence, or guide sequence had been designed complementary to 5′-end bla mRNA sequence with the required 5′-end tag. Good result is the gene get silenced which shows promise to another novel silencing systems in bacterium and archaea.


CRISPR/Cmr with RNA as its target is just one subtype of CRISPR/Cas system. Other types target DNA. 

If we took a deep look at the general features of all the systems, and compare them with eukaryotic RNAi side-by-side,[4] there are still lots of questions remained unsolved, and mechanisms left mysterious.

How the invading sequence get integrated into CRISPR loci?

CRISPR/Cas system is adaptive immune system, not innate. Bacteria and archaea are not born with it, and they need immune stimulation at first to gain a short of sequence from invading virus or plasmids. And it is the short foreign sequence that gets integrated into CRISPR loci between two short repeat sequences and enables crRNA to bind with RNA/DNA.

But what is mechanisms of the acquisition? Unknown[4].

How to discriminate self or invading?

For CRISPR/Cas system that target DNA, the 5′-end tag (in short repeat region) is critical for distinguishing self from non-self. If the 5′-end tag mismatches the invading DNA, the invaders must die. If the tag precisely matches foreign DNA, it is considered as the host CRISPR locus itself and does not “attack”. What about  CRISPR/Cmr? 

To discern the function of 5′-end tag in CRISPR/Cmr targeting RNA. Three disturbance experiments are conducted. When the 5′-end tag is totally deleted, substituted by other types of sequences (one is precisely complementary to itself, two are with just first one or two bases complementary to itself), these three new tags are no more original 5′-end tag leading to the silencing effect disappear, just as expected. Thus it can be concluded that 5′-end tag is sufficient and critical for RNA silencing. 

But an interesting experiment leaves the tag’s function more confusing[3]. If the target transcript sequence is complementary to the tag, even though the target is known to be CRISPR hosts sequence, the RNA cleavage is not prevented. Just like shown in the right half figure, the target sequence cannot escape from being killed even it is complementary to the 5′-end tag. Thus 5′-end seems not to be the key commander in discrimination, or there are other molecules hold the key? At least, another unknown issue. 

What can Synbio do?

A DNA silencing systems in bacteria, and novel RNA silencing system in archaea!!! It leaves up to you.


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  1. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D. A., et al. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science (New York, N.Y.), 315(5819), 1709-12. doi:10.1126/science.1138140
  2. van der Oost, J., & Brouns, S. J. J. (2009). RNAi: prokaryotes get in on the act. Cell, 139(5), 863-5. doi:10.1016/j.cell.2009.11.018
  3. Hale, C. R., Majumdar, S., Elmore, J., Pfister, N., Compton, M., Olson, S., Resch, A. M., et al. (2012). Essential Features and Rational Design of CRISPR RNAs that Function with the Cas RAMP Module Complex to Cleave RNAs. Molecular Cell, 1-11. Elsevier Inc. doi:10.1016/j.molcel.2011.10.023
  4. Wiedenheft, B., Sternberg, S. H., & Doudna, J. a. (2012). RNA-guided genetic silencing systems in bacteria and archaea. Nature, 482(7385), 331-338. doi:10.1038/nature10886

Copyright: The attached figures belong to publications with reference number, respectively.

Uploaded and Uploading to SynBio world

Synthetic Biology, an emerging interdisciplinary field, is building up a bridge to get biologists and engineers collaborated widely, science and technology merged tightly, dreams and reality synchronized fully. There are countless fascinating projects and ideas in SynBio world which are supposed to have great implication to our real world and of course, one cannot depict them in details. In addition, it is hard and also does not make sense to trace back the exact date when scientists managed to build novel bio-pathways, when living cells were hijacked by human design, and when some synthetic biology began to appear.

Here I’ll simply follow reviews from 2004 to 2011 published in Nature, and it is amazing to find that these reviews mark out the milestones of SynBio, and introduce what have been uploaded to SynBio.

Wall, M. E., Hlavacek, W. S., & Savageau, M. a. (2004). Design of gene circuits: lessons from bacteria. Nature reviews. Genetics, 5(1), 34-42.
Mukherji, S., & van Oudenaarden, A. (2009). Synthetic biology: understanding biological design from synthetic circuits. Nature reviews. Genetics, 10(12), 859-71.
Purnick, P. E. M., & Weiss, R. (2009). The second wave of synthetic biology: from modules to systems. Nature reviews. Molecular cell biology, 10(6), 410-22.
Khalil, A. S., & Collins, J. J. (2010). Synthetic biology: applications come of age. Nature reviews. Genetics, 11(5), 367-79.
Weber, W., & Fussenegger, M. (2011). Emerging biomedical applications of synthetic biology. Nature Reviews Genetics, 13(1), 21-35.
computer parallel to life

The key words highlighted in the title precisely point out what have been accomplished in SynBio world. From the very beginning, SynBio has regarded living cells as machines. In silico, we have physical layer (electric resistance), Boolean Gates, Modules, Computers, and finally Networks. In vivo, we have Genes, Biochemical Reactions, Bio-pathways, Cell, and finally Tissues (furthermore individual bodies), respectively [1]. Systems biology shares such a point of view, however, the distinguishing goal of Synthetic biology, is to re-engineer organisms, which brings to the 2011 review — biomedical applications. More comments and typical examples are available in Synthetic Biology is on its Way to Treating Human Disease from Bio_2.0 by Eric.

Here I really want to highlight a “smart-drug” that kills targeted cancer cells, an application based on microRNA though it is 4-month “old” story. The device can first recognize HeLa cells and furthermore simultaneously induce apoptosis.

miRNA logic device

This project by Xie, etc. aims to construct a classifier marked within the dashed line shown in part D. [2] Five signals, native microRNAs in cells, are used. Two are highly and  exclusively expressed in HeLa cells, while three are in low expression. The mission of the device is to precisely recognize them so that regular cells and HeLa cells can be classified and then produce hBAX protein to induce cell death (shown in  part A).

The device runs like this, and vice-versa.

Hela_high_1 = 1; Hela_high_2 = 1;

Hela_low_1 = 0; Hela_low_2 =  0; Hela_low_3 = 0.

The output = 1. Apoptosis ON. Hela-cell DEATH.

Specifically, part B shows the logic gates for miRNA highly-expressed in HeLa cells, while part C demonstrates those in low expression. The logic gates dealing with miRNA-hela-high and miRNA-hela-low are different. For miRNA-hela-high, Xie, etc. designed a sensor motif for Hela-high markers comprising a “double-inversion” module that allows output expression only if the marker is present at/above its level in Hela cells, otherwise represses the output if the marker’s level is low. Researchers need “double-inversion” module, is because among the 2^5=32 validation experiments, some exceptions come across the undesirable leakiness problem, in other words, when the miRNA-hela-high is set NULL, the output expression is undesirably high. The “double-inversion” module manages to deal with leakiness problem after it’s been incorporated.

Logic_device. After "computation", the delivered miRNA-mRNA device will function to induce cell death or leave it alone.

The research itself builds up a example or model. Once you can design miRNA-logic-gates to recognize and exclusively kill HeLa cells, the most widely used immortal cell line in bio-researches, you can apply the same strategy dealing with other cancer cells, which is a potential bio-medical application.

Though it’ s promising,  I guess the next step of the research group is to develop a “capsule” to deliver the miRNA-logic-gates into patients’ cells. As the research still lies in cell-level, not individual-body level, which means it has long way to go.

The rest of the miRNA-device is self-explaining. In addition, more general information about miRNA-mediated mRNA degradation can be available in this animation on Nature.

Synthetic biology is sometimes misunderstood as a duplicate of systems biology, which takes some scientists to clarify the difference and point out the synergy. [3] Now synthetic biology is no more simply a shadow of systems biology, partly because of the accomplished bio-applications.

Auxin by Imperial College London
Auxin by Imperial College London. The green light (expressed GFP) show the position of E.coli inside plant roots.

Applications in real world, win iGEM_2011 as well. In the past, most iGEM teams come up with new designs, new biobricks, new modules, but they leave the applications to “future work”. Awkward. However in 2011, “Auxin” by Imperial College London, “Make it or Break it” by University of Washington, managed to achieve promising application which shows great implications for the future. Of course you can imagine engineered E.coli stimulate plant root growth and probably address the global soil erosion issues, but you can never expect that this idea can really be put into solid ground and into practice NOW! [4] A deep and remarkable thoughts towards iGEM_2011 and applications, by Rob Carlson, is highly recommended.

I keep wondering, what is the next big move, the very next milestone of Synthetic Biology? Could it be de novo direct synthesis of full-genome, Biofuel, or Bio-computers? That leaves to you.

At last, if you want to know more about synthetic biology, this free on-line “textbook” on WikiGenes, is suggested.

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[1]  Andrianantoandro, E., Basu, S., Karig, D. K., & Weiss, R. (2006). Synthetic biology: new engineering rules for an emerging discipline. Molecular systems biology, 2, 2006.0028.doi:10.1038/msb4100073

[2]  Xie, Z., Wroblewska, L., Prochazka, L., Weiss, R., & Benenson, Y. (2011). Multi-Input RNAi-Based Logic Circuit for Identification of Specific Cancer Cells.Science, 333(6047), 1307-1311. doi:10.1126/science.1205527

[3] Smolke, C. D., & Silver, P. a. (2011). Informing biological design by integration of systems and synthetic biology. Cell, 144(6), 855-9. Elsevier Inc. doi:10.1016/j.cell.2011.02.020