Team:Strasbourg/Scientific Background

iGEM

Aptamers

Aptamers are oligonucleotides (RNA, DNA, XNA) discovered in the late 20th century [1], which have a strong affinity for a specific target molecule called the ligand. Depending on the aptamer sequence, the ligand could be small molecules, peptides, proteins, cells, and tissues. Aptamer recognition of specific ligand induces its structural change. They naturally exist in bacterial riboswitches as regulatory elements of gene expression (transcriptional or translational) for quick response to environment changes [2].

These aptamers can be engineered in-vitro by selection from a large library of a random sequence of DNA or RNA, for example using SELEX (Systematic Evolution of Ligands by EXponential enrichment) to recognize specifically ligands. They can be used in a wide range of research as fundamental biology, diagnostic, and therapeutic applications (Fig.1).

Figure 1: Gli4 aptamer from Aptagen.com [3].

Ribozyme

In 1980 Tom Cech and Sidney Altman (Nobel Prize in Chemistry, 1989) discovered RNA molecules called ribozyme that are capable of catalyzing chemical reaction as enzymes. Originally, ribozymes are applicated to mature non-translatable RNA as tRNA. Today ribozymes such as aptazymes are used in biotechnology [4]. Their capacity of cleavage has a potential therapeutic application for the inactivation of deleterious genes and the repair of mutated genes involved in many disease states.

Figure 2: Sequence and secondary structure of the natural occurring env-9 motif. The pseudoknots Pk1 and Pk2 are highlighted in green and blue, respectively. The inactivating mutation is highlighted in yellow. The cleavage site is indicated with a grey arrow [5].

iGEM Strasbourg

iGEM Strasbourg 2019 develops a new specific and flexible molecular detection system. Many detection gadgets are based on antibodies which are expensive because of the use of animal for their production and the use of mass spectrometry for their quality analysis. Aptamers are potential concurrent to antibodies, for many reasons [6]:

  • Contrary to antibodies, aptamers can be selected to detect non-immunogenic small molecules and metabolites as well as compounds that are toxic to cells.
  • Traditionally, antibody development requires 6 months of work and animal use. Classical aptamer selection is an in vitro process that is typically completed in two to three months.
  • Aptamer complexes have been shown to cross the blood-brain barrier and even enter cells.
  • The use of DNA or RNA aptamers eliminates potential interference from endogenous antibodies.
  • Aptamers are chemically synthesized, so the aptamer sequence and its modification(s) are the only information required to maintain long-term access to the aptamer that was originally produced and validated.

This year, we would like to use aptamer specific targeting and ribozyme auto-catalytic properties for modulating gene expression in E. coli bacterial hybrid system [7]. This is possible with aptazymes developed by Jorg Hartig [5]. Two different types of aptazyme exist: guanine switch-on (auto-cleavage in presence of ligand) (Fig.3) and theophylline switch-off (auto-cleavage in absence of ligand).

Figure 3: Aptazyme switch-on

Thanks to aptazymes flexibility, our detection system should be fast and portable for easy on-site use, as well as versatile to adapt to any molecule. To identify and develop the best molecular platform for aptazyme two constructs will be tested. If you want to discover more about our constructs, please check our scientific strategy

References

  1. Ellington, A. D., Szostak, J. W. 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346: 818-822.
  2. Ronald Breaker and Evgeny Nudler discovered a nucleic acid-based genetic regulatory element, 2002.
  3. Gonzalez, S., et. al. 2014. Aptamer Binding to Celiac Disease-Triggering Hydrophobic Proteins A Sensitive Gluten Detection Approach. Analytical Chemistry 86: 2733-39.
  4. Ruffner, D. E. 2001. Ribozymes in Biotechnology. In eLS, (Ed.).
  5. Stifel, J., Spöring, M., Hartig, J. S. 2019. Expanding the toolbox of synthetic riboswitches with guanine-dependent aptazymes. Synthetic Biology 4.
  6. https://www.basepairbio.com/aptamers-vs-antibodies/. [Accessed 10/20/2019]
  7. Berry, K. E., Hochschild, A. 2018. A bacterial three-hybrid assay detects Escherichia coli Hfq-sRNA interactions in vivo. Nucleic Acids Research 46.
  8. Daines, D. A., Granger-Schnarr, M., Dimitrova, M., Silver, R. P. 2002. Use of LexA-based system to identify protein-protein interactions in vivo. Methods Enzymol 358:153–161.