Introduction

Organosilicons are organometallic compounds that consist of carbon-silicon bonds. They are comparable to their corresponding organic analogs but differ in their intrinsic properties. These differences, especially the chemical properties of silicon and its bond formation tendencies, have a significant impact on their bioavailability and their application in medicine. Recent publications cluster their unique features into three categories: The first category comprises the chemical properties of silicon bonds. Typically, silicon forms longer bonds at different angles, which leads to diverse ring conformations and thus, alterations in reactivity. Furthermore, its preference to form single bonds leads to chemical compounds that have a higher intrinsic stability than their carbon analogs. The second category represents the bioavailability of organosilicons. They are more likely to overcome the membrane barrier of cells as they are more lipophilic compared to their respective carbon counterparts. The third - and most important - category deals with the medical application of these compounds. Due to their aforementioned tendency to form single rather than double or triple bonds, they display a viable source for stable pharmaceuticals, which are inaccessible as carbon-based molecules. Additionally, the more electropositive nature of silicon facilitates hydrogen bond formation and conveniently increases the acidity of the compounds. As a result, organosilicons address the major issue in the synthesis of bioactive pharmaceuticals, the design of pro-drugs, as well as a safe medicine with a genuine biomedical benefit. Thus, their main advantage is to operate as pro-drugs due to their thermodynamic stability, but aqueous and acidic instability. On top, as we know so far, silicon is nonhazardous by itself, which makes it a valuable source for further biomedical research.

Our Idea

As a proof-of-principle, we wanted to show and harness the potential of organosilicon-forming proteins. Therefore, we used a previously engineered cytochrome c enzyme and coupled organosilicon-production directly to a reporter expression. Thereby, we were focusing on a small molecule-sensing riboswitch as proposed underlying mechanismHenkin.2008. This riboswitch was designed in silico using the MAWS software, that was provided by the iGEM Team Heidelberg 2015. In a step-by-step approach, we wanted to produce an organosilicon which, in the end, could be tested with the designed riboswitch to express the NanoLuc reporter (Promega). The NanoLuc is currently the most sensitive luciferase available, and is able to show us a significant output despite using only a small amount of substrate.

Experimental procedures

Design and cloning of the riboswitch and cytochrome c constructs

The educts for the organosilicon synthesis were commercially available in the case of compound dimethyl(phenyl)silane (2) and ethyl 2-diazopropanoate (5) or were custom synthesized by Fabian Ebner (Greb group, ACI Heidelberg, Germany) in the case of compound 4-(dimethylsilyl)aniline (1). The corresponding riboswitch was designed accordingly using the MAWS software developed by the iGEM Team Heidelberg 2015. To obtain the riboswitch sequence, the chemical structure of the desired product was in silico aligned to randomly generated RNA sequences which were scored according to their ability to form hydrogen bonds with the product. The most favorable sequence (BBa_K2398555) was ordered as oligos which were annealed in a single-cycle Touch-Down PCR, diminishing the temperature by 0.1°C x sec^-1 95°C to 10°C. The sequence was ordered as oligos and not as gBlock to ensure overhangs of a specific length and sequence at the 5’ and 3’ ends. The vector and reporter were amplified via PCR and purified by gel extraction (Qiagen). The final plasmid was assembled by using an equimolar concentration of the vector, reporter, and the riboswitch in a golden gate reaction. The plasmid was amplified after transformation in DH10beta cells and purified via plasmid purification (Qiagen). To make organosilicon production more approachable for other iGEM Teams, we codon optimized the wild-type cytochrome c derived from Rhodotermus marinus and cloned it into the pSB1C3 vector. We are proud to present you this part as our desired best basic part. For cloning purposes, we ordered the optimized version as gBlock containing a biobrick prefix and suffix for restriction cloning. We were using this part as a triple mutant, created by F. ArnoldKan.2016, that showed a higher activity towards our substrates. For more characterization of this part you can find the documentation here.

Riboswitch binding assay

Results

Synthesis of the organosilicon compounds

Outlook

The increase in enzyme activity upon addition of the specific riboswitch activator was determined in relation to the enzyme activity in presence of the initial substrate. This is demonstrated in Fig. y that shows the 1.5 fold enzyme activity when using the newly synthesized substrate. Overall, these results show the proof-of-principle of the application of organosilicons in the use of riboswitches. Moreover, it presents the promising opportunity to further subject small molecule-sensing riboswitches to an enzyme PACE approach (PREDCEL) for the enhancement of downstream reactions such as gene III activity to facilitate phage propagation.