Greatly enhanced detection of a volatile ligand at femtomolar levels using bioluminescence

resonance energy transfer (BRET)

by H. Dacres et al.Biosensors and Bioelectronics 29 (2011), 119–124

doi:10.1016/j.bios.2011.08.004Impact Factor-6.409

Diksha JainBO15MTECH11004Mid-Sem Assignment

Aim• To develop a GPCR-based odorant biosensor with improved

sensitivity and limits of detection• Successful engineering of an odorant biosensor would

represent proof of concept for a new, biomimetic, class of electronic nose sensors with a number of potential applications including explosive detection, quality control of food and beverage production and clinical diagnosis as well as drug discovery

Fig: Flow chart of a typical Biosensor

GPCR• ODR-10 odorant receptor (odr, or ODorant-Response

mutants) from Caenorhabditis elegans was choosen as the initial test of this concept because it is a well-characterised chemoreceptor and potentially representative of several hundred other nematode chemoreceptors

• ODR-10 is a predicted seven transmembrane domain receptor of the G protein–coupled superfamily, which is specifically present only in nematodes

• ODR-10 is exclusively activated by diacetyl (2,3- butanedione)• A point mutation ky32 (H110Y), which is a G to A transition on

the non-coding strand of odr-10 gene in the third predicted membrane-spanning domain of the protein (IC3), renders ODR-10 receptor non-functional

Method1. ODR-10 chimaeras (OGOR) were constructed by inserting

GFP2 (green fluorescent protein , BRET acceptor) between amino acids 240–241, which is the third intracellular loop (IC3) and RLuc (Renilla luciferase protein, BRET donor) at the C-terminus of ODR-10

2. OCOY, the non-functional control version of OGOR was constructed, by introducing a point mutation ky32 (H110Y). CFP (cyan fluorescent protein), was inserted at the IC3 and YFP (yellow fluorescent protein) at the C-terminus of the ODR-10

Fig: OGOR

Fig: Principle of bioluminescence resonance energy transfer (BRET) in ODR-10 receptor constructs fused to Renilla luciferase (RLuc) and green fluorescent protein (GFP2). GFP2 is inserted in the third intracellular loop of ODR-10 and RLuc at the C-terminus (OGOR). Diacetyl binding causes a conformational change in the OGOR biosensor resulting in an increase in distance, or a change in the orientation of dipole moments, between the BRET2 components. Clz400a = Coelenterazine 400a acts as a substrate for Rluc causing bioluminescence

3. OGOR was expressed in the S. cerevisiae strain, INVsc1, a diploid strain, which does not express an intrinsic GPCR signaling pathway i.e. a ‘null background’ for nematode OR (Olfactory Receptor) studies

4. A cell-free preparation of membranes was used because it has the additional advantage of eliminating the potential barrier to free movement of some ligands presented by the yeast cell wall

5. OGOR expression was confirmed by immuno-blotting . Analysis was carried out using goat anti-ODR-10. Immuno-detection used streptavidin-biotinylated horseradish peroxidise complex with 4- chloro 1-naphthol as substrate.

6. OGOR localization in plasma membrane and intracellularly was confirmed by Fluorescence Microscopy. Samples were excited at 488 nm and images were collected at 510 nm for GFP2 emission and 660 nm for Evans blue emission

Fig: Expression of olfactory receptor ODR-10 and OGOR in S. cerevisiae (INVsc1) cells. Immuno-blotting analysis of isolated membranes from INVsc1 cells expressing untagged ODR-10 (Lane 1) and INVsc1 cells expressing BRET2 tagged ODR-10, OGOR (Lane 2)

Fig: Visualization of expressed OGOR in INVsc1 cell. Top and bottom panel images left to right- 1) INVsc1 cells stained with Evans blue dye – a plasma membrane specific dye; 2) GFP2 signal; 3) Overlay of two previous images showing plasma membrane localisation. Top panel scale bar = 20 µm, bottom panel scale bar = 2 µm

7. In-vivo control exp: To test that the ligand binding properties of the OGOR sensor accurately reflect ODR-10 function in vivo. Non-functional OGOR mutant, OCOY was expressed in yeast cells• Upon diacetyl exposure to OCOY, a 4.1% drop in BRET2

signal was observed, which was not significantly different (P = 0.379) from the control (water). In vivo, this single amino acid (H110Y) change ablates nematode chemotaxis to diacetyl• Upon addition of 1 µM diacetyl to OGOR, there was a 32%

decrease in BRET2 signal• This observation indicates that the diacetyl-induced change

in BRET2 signal requires the same ligand binding structure as is present in native ODR-10

Fig: BRET2 signal from OGOR and OCOY (OGOR mutant (H110Y)), following a 45min incubation with 1 µM diacetyl in water and a water only control (NS denotes no significant difference; P ≥ 0.05 compared to water)

8. Qualitative BRET2 assay-

Fig: BRET2 responses of OGOR to addition of a range of volatile and non-volatile compounds at 1 µM (white bars) and 1 nM (grey bars) concentrations. ** denotes significance at P ≤ 0.01 compared to water. Patterned bar represents response to water

• A test mixture containing 10 nM citric acid and10 nM butanediol did not elicit a BRET2 response significantly different from water alone (P = 0.3011), however, addition of 10 nM diacetyl to this mixture induced a highly statistically significant response (P = 0.0041) which was not statistically different from the response to diacetyl in water (P = 0.7514)

• These results confirm that the isolated BRET2 tagged ODR-10 receptor retains the ODR-10 receptor’s in vivo and in vitro ability to specifically bind diacetyl• Also, ODR-10 is solely responsible for chemotaxis to diacetyl

9. Quantitative BRET2 assay-• In vitro OGOR response to diacetyl is dose dependent, with

a linear range spanning nine log units, from 10−19 to 10−10M. The calculated EC50 value is 3.55 fM for diacetyl, which is equivalent to 0.31 parts per quadrillion (ppq) (w/v)• In vivo response to diacetyl is effectively ablated over all

concentrations in OCOY, thus in vitro OGOR response to diacetyl is therefore consistent with the response of ODR-10 in the whole organism

Fig: Diacetyl concentration dependence of the OGOR BRET2 response

TR-FRET • BRET offers the simplicity of receptor labeling performed by

bioengineering techniques. This is a strong advantage but also a disadvantage since it is impossible to distinguish the receptors targeted to the surface or trapped inside the cell. All the donors are derived from Rluc, the development of a different and smaller luminescent donor is much required

• With an appropriate flow arrangement, it will be possible to measure ligand transduction in a better way with time resolution i.e. TR-FRET

• TR-FRET displays an even larger panel of tools for receptor labeling. Covalent labeling techniques offer some advantages with much smaller tags as compared to BRET

• TR-FRET based on fluorescent ligands is the only technique that can be applied to wild-type receptors expressed in a native context

Result & Conclusion• The biosensor design described in this paper is more than three

orders of magnitude more sensitive than the most sensitive biosensor reported yet in the literature for monitoring diacetyl binding

• The superior sensitivity, of the in vitro GPCR-BRET2 assay and retention of in vivo receptor characteristics, is the result of combining yeast expression with a cell-free assay and BRET2 detection

• The development of biosensor technology incorporating nematode ORs provides the basis for a bioelectronic nose mimicking the invertebrate olfactory system. Such a device could be used to identify and monitor a spectrum of odorants in real-time with much higher selectivity and sensitivity than present electronic devices