Team:ZJU-China/project.htm

This year, the ZJU-China iGEM team aims to design and realize a tunable RNA scaffold to accelerate biosynthesis pathways and turn their on and off. As one of the most vital biomacromolecules, RNA plays a crucial role not only in coding process, but also in non-coding one. RNA scaffold is designed to colocalize enzymes through interactions between binding domains on the scaffold and target peptides fused to each enzyme in engineered biosynthesis pathways in vivo, which may suffered from low efficiency of production caused by relative lack of spatial organization of non-homologous enzymes. The scaffold allows efficient channeling of substrates to products over several enzymatic steps by limiting the diffusion of intermediates thus providing a bright future for solving the problem. Meanwhile, we plan to add an aptamer structure on RNA scaffold as a riboswitch to regulate biosynthesis pathways by micromolecular ligands. Then we can control the all-or-none binding relationship between the enzymes and RNA scaffold by whether the special ligands are presented or not.

In previous work, FA and FB are used to indicate the efficiency of riboscaffold. In order to further prove the function of riboscaffold, we plan to substitute FA, FB with functional enzymes or protein substrates like ferredoxin in hydrogen producing pathway respectively.

Considering the availability of material and abundant parts distributed by iGEM, we search the 2012 kit plate1-5 to find optimal pathways. After a pre-selection, six pathways are on candidate list. For sake of experimental feasibility, we perform a further selection based on several caritas as follows:

1. Product is easy to detect and measure;

2. Substrate is easy to get;

3. Product is beneficial to human;

4. The length of amino acid sequence of enzyme is optimal to be fusion protein;

5. Two proteins involved in the basic pathway.

Candidate list:

1. Salicylate pathway (Group: iGEM2006_MIT)

Assessment:

The characterization method of gas chromatography is difficult to perform. First, what can be analyzed is methyl salicylate production, that is to say, another enzyme should be co-transformed to E.coli too, which will increase cell’s burden and reduce the ratio of successful co-transformation. Second, it is not convenient for us to borrow the relative machine.

2. Pyocyanin pathway (Group: iGEM2007_Glasgow)

Assessment:

Through there are exactly two enzymes involved in this pathway, but the source of material, phenazine-1-carboxylic acid (PCA), is not mentioned. And it not easy to measure the amount of pyocyanin.

3. Lycopene pathway (Group: iGEM2009_Cambridge)

Assessment:

Lycopene is visible red and its substrate, FPP, is colorless. So measurement is quite feasible. But there are at least three proteins in this pathway, which will increase the burden of cell. But in future work, we could have a try.

4. Holo- α -phycoerythrocyanin pathway (Group: iGEM2004_UTAustin)

Assessment:

Heme is metabolic product of E.coli and Holo-α-phycoerythrocyanin is blue. But at least 5 proteins should be expressed in E.coli.

5. BPA degradation pathway (Group: iGEM2008_University_of_Alberta)

Assessment:

Bisphenol A is degraded by BisdA and BisdB. But BPA is toxic to cells.

6. IAM pathway (Group: iGEM2011_Imperial)

Assessment:

Five pathways described above all have some drawbacks, finally, only one pathway left, IAM pathway. The two-step IAM pathway generates indole-3-acetic acid (IAA) from the precursor tryptophan. IAA tryptophan monooxygenase (IaaM) catalyses the oxidative carboxylation of L-tryptophan to indole-3-acetamide, which is hydrolysed to IAA and ammonia by indoleacetamide hydrolase (IaaH).

Applications of RNA Scaffold & Aptamers

1. RNA aptamers take place of fluorescent proteins

Some RNA aptamers can bind fluorophores, such as 4-hydroxybenzlidene imidazolinone (HBI), 3,5-dimethoxy-4-hydroxybenzylidene imidazolinone (DMHBI), 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), resembling the fluorophore in GFP, and then these RNA-fluorophore complexes enable to emit different colors of fluorescence comparable in brightness with fluorescent proteins.

These RNA-fluorophore complexes could be used to tag RNAs in living cells to reveal the intracellular dynamics of RNA, including RNA-RNA and RNA-protein interactions.

[Reference: Jeremy S. Paige, Karen Y. Wu, Samie R. Jaffrey, RNA Mimics of Green Fluorescent Protein science, 2011 vol 333, 642-646]

2. kinetic investigation of RNA hybridizations and foldings

By introducing fluorophores like 1-ethynylpyrene into the 2-position of RNA adenosine, through an intermolecular interaction of the pyrene residues in twofold labelled RNA, single and double strands can be distinguished by their fluorescence spectrum changes.

With this fluorescence shift, one can distinguish between single-stranded and double-stranded RNA during thermal denaturation. This behavior could be used for the time resolved investigation of RNA hybridizations and folding by fluorescence spectroscopy.

[Reference: Josef Wachtveitlb, Joachim W. Engels, ect. RNA as scaffold for pyrene excited complexes, Bioorganic & Medicinal Chemistry 16 (2008) 19-26]

3. Medicine & health

To date, many groups have successfully identifi ed aptamers with a variety of functions, including inhibitory and decoy-like aptamers, regulatable aptamers, multivalent/agonistic aptamers, and aptamers that act as delivery vehicles. Each of these classes of aptamers has potential applications in therapeutics and/or diagnostics.

Inhibitory aptamers：The most extensively characterized inhibitory aptamer is the RNA aptamer that targets VEGF. This aptamer was approved by the FDA in December 2004, for the treatment of wet age-related macular degeneration (AMD)

Decoy-like aptamers：By mimicking the target sequence of the proteins, aptamers can act as decoys to inhibit binding of transcriptional factors such as HIV-tat, NF-κB, and E2F to their cognate sequences on DNA and thus prevent transcription of target genes and may result in powerful therapeutics for treating many human pathologies

Multivalent aptamers: A bivalent aptamer targeting HIV has also been described and consists of 2 separate RNA aptamers that bind to 2 distinct stem-loop structures within the HIV 5′UTR: the HIV-1 TAR element and the dimerization initiation site. Similarly, bivalent aptamers targeting thrombin have been engineered as a way to increase the avidity of the aptamer for its target and enhance the anticoagulation effect

Aptamers as delivery tools: Several groups have reported linking siRNAs to aptamers as a way to specifi cally deliver siRNAs to target cells. Aptamers are also being utilized to deliver toxins, radioisotopes, and chemotherapeutic agents encapsulated in nanoparticles.

[Reference: Kristina W. Thiel and Paloma H. Giangrande, Therapeutic Applications of DNA and RNA Aptamers. Oligonucleotides, 2009, Volume 19, Number 3, 209-222]

4. Regular of gene expression

Aptamers are small oligonucleic acid molecules that can be selected in vitro against nearly any target of choice. And they often show remarkable binding affinity and specificity, and consequently have a huge potential for application. One of their usages is to play a role in activating gene expression.

Some RNA aptamers can specifically bind some transcriptional regulator. For example, people have selected one RNA aptamer that can bind TetR, which usually binds on operator sequence and repress gene expression. So once the RNA aptamer binds to the transcriptional regulator, the targeting gene-expression is activated.

[Reference: Anke Hunsicker, Markus Steber, ect. An RNA Aptamer that Induces Transcription, Chemistry & Biology, 2009,Volume 16, Issue 2, 173–180]