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

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