Team:Stanford-Brown/SB16 BioSensor IRES

Introduction

A fully functional sensor needs both a way to recognize the small molecule it’s supposed to be sensing (a recognition element) and a detectable signal that reliably results from a positive sensing interaction (an expression platform).

An aptamer is defined by Sun et al as a “nucleic acid with high specificity and affinity for its target”.[1] As such, aptamers can be used to build highly discriminating recognition elements for their target ligands. However, alone this is not enough -- molecular conformational changes are no easier to detect than the target small molecules themselves.

Inspired by a 2011 paper from a group at Ehime University, we sought to connect the recognition functionality of specific aptamers with in vitro expression of a particular reporter gene (for our constructs we used GFP). To do this, we synthesized an “IRES sensor” construct (see picture below). IRES stands for internal ribosome entry site. It does exactly what the name implies: allows transcription and thus translation to happen. The key for our purposes is that IRES has a particular active 3-D confirmation: if the IRES is folded correctly it facilitates protein translation, if not it doesn’t so no translation will occur.

Ogawa’s work showed that disrupting a particular region of the IRES (shown here in blue) by binding it with its complement, anti-IRES (aIRES, shown here in pink), rendered the entire IRES sequence inactive and unable to translate. In the “off” state when the aptamer is unbound, IRES is bound by a-IRES and no reporter gene expression is observed. In this state the complement to aIRES, anti-anti-IRES (aaIRES, shown here in red), is trapped in an inaccessible hairpin by the end of the aptamer and the optimized modulator region.

When the target molecule is introduced, it binds to the aptamer. As a result, the aptamer changes conformation, and releases the aaIRES sequence. The released aaIRES sequence binds to aIRES (out-competing IRES), freeing the IRES complex to refold into its active conformation. Once IRES is correctly folded, this allows the sensor to translate reporter protein. The plasmid we created to accomplish this is shown in Figure 1, with the resulting molecules pictured in Figure 2.

Figures

Figure 1: Annotated Geneious Map of the IRES system.

This construct contains the iGEM standard prefix, a constitutive promoter (BBa_J23119), a 5’ loop that Ogawa found to be helpful in stabilizing the construct, a “modulator region” that determines how much of the aptamer is bound to aaIRES, the aptamer of interest, aIRES, a naturally occurring viral IRES, the translation start site at the very end of it, and finally the GFP coding sequence.

Figure 2: Schematic of the IRES sensor system. Modified from figure 5, “Rational Design of Artificial Riboswitches…” (Ogawa, 2011)

Figure 2 shows how the sensor works at the molecular level. On the left (OFF): the aptamer is unbound, and the functional IRES (blue) is bound to the aIRES (red). Because of this, the functional 3-D confirmation of the IRES complex is disrupted, blocking the translation start site, preventing transcription of the reporter gene. On the right (ON): aIRES (red) is bound in this case to aaIRES (pink) rather than IRES (blue). This allows the functional IRES complex to form, and translation of the reporter protein to occur.

We were not able to replicate the IRES sensor behavior of the Ogawa paper. At first we realized this was because we had selected a constitutive promoter that was incompatible with our in vitro translation system. Even after expressing our IRES sensor with forward promoters that incorporated T7 however, we still did not see any GFP expression even in conditions with high concentrations of theophylline (the target molecule for our sample aptamer.) We next postulated that the observed inactivity may be due to the fact that our engineered plasmid completely lacked a gap between the translation start site and the AUG start codon of GFP. Accordingly we linearized the plasmid at the point where the TSS meets GFP, amplified this template with primers that incorporated a 6nt gap region, and performed blunt-end ligation to re circularize the plasmid. Post recircularization, we re-transfected E Coli with the gap-containing plasmid, and were still unable to observe differential GFP expression in response to different concentrations of theophylline.