A Dual-Color Far-Red to Near-Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase (Fluc). However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual-color, far-red to near-infrared (nIR) emitting analogue of beetle luciferin, which, akin to natural luciferin, exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima up to λmax=706 nm with different Fluc mutants. This emission is the most red-shifted bioluminescence reported without using a resonance energy transfer acceptor. This improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging.

Over-expression, protein purification by NiNTA chromatography, SDS-PAGE and protein assays were carried out as detailed previously. [1] Spectrofluorometry. Absorbance and fluorescence spectra were acquired in Thermo Flash (Thermo Fisher Scientific, MA, USA) by 1:10 dilution of free acids of luciferin (1), our analogue (6) and the Maki red analogue (5) in TEM buffer at pH 7.8 at room temperature. See Supplementary Figure S2 legend for details. To measure pH dependence of fluorescence spectra compounds were diluted in TEM buffers prepared between pH 6-9.
In vitro characterisation of bioluminescence properties. In vitro data was obtained by saponifying the esters using Porcine Liver Esterase (PLE -Sigma Aldrich, MO, USA) for at least 15 min prior to the addition of luciferase, without isolation of the free acid. TEM buffer (100 mM Tris-acetate, 2 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM magnesium sulfate (MgSO 4 )) was pH adjusted at room temperature (RT) with 1-10 M sodium hydroxide or 6-10 M acetic acid. Bioluminescence spectra were acquired using a Cary Eclipse spectrophotometer (Agilent Technologies, CA, USA) using PMT gains of 800-1000 V and emission slits of 20 nm and 100 ms gating. Signal averaging was carried out if required ( Figure 2) and spectra were corrected for variation in photomultiplier tube spectral sensitivity by calibrating with lucifer yellow (Sigma, MA, USA) and 2- styryl]-1methylpyridinium iodide (ASP+) (Sigma, MA, USA) dyes, and using the known corrected spectra from Molecular Probes.  Table 1: To initiate bioluminescence, 50 µL of 0.1 mg/mL 1 or 1 mg/mL of 5 or 6 and 20 µL 20mM ATP was added to 20 µL 7µM solutions of Fluc in cold TEM buffer.
Specific activities of enzymes were calculated by integrating light emission from 1 mM substrates, 5 mM ATP and 0.5 µM Fluc in 100 uL TEM at room temperature and using no filter for 2 min in the Photon imager device (Biospace Labs, Paris, France). FWHM: bioluminescence Full-width half-maximum. Details as in Table 2. Error +/-2 nm. imager (Caliper, USA) and kinetic parameters were calculated using the Hanes-Woolf plot. [2] Specific activities of 1, 5 and 6 esters with purified enzymes: to capture specific activities of enzymes with substrates, 40 µL 0.5 µM x5 Fluc in 5 mM TEM buffered ATP was added to saponified esters and light emission was captured for 2s using the IVIS Imager.
Production of human retrovirus, transduction and maintenance of cell lines. Retroviral vector was generated by co-transfecting 293T cells with vector plasmid, along with RDF plasmid which supplied the RD114 envelope as described previously. [3] Cell lines were either purchased from ATCC, or provided by the UCL Cancer Institute cell bank. Flow cytometry was carried out using anti-Myc primary (Invitrogen Life Technologies, CA, USA) and anti-Fc DyeLight 647 (Jackson ImmunoResearch, PA, USA) secondary conjugated antibodies in a CyAn instrument (Beckman Coulter, Inc., Brea, CA, USA). The percentage of stained cells (expressing the Myc tag) was used to ascertain the percentage transduction. The mean fluorescence intensity of the DY 647 antibody was used as a measure of expression level of the gene cassette. Transduction of 3 x 10 5 Raji cells with 1.5 mL of viral supernatant was carried out by using retronectin (Takara Bio Inc., Shiga, Japan) according to manufacturer's instructions. Cells were cultured at 37 °C with 5% carbon dioxide in RPMI1640 with 10% foetal calf serum (FCS) and glutamine (GlutaMAX TM , Invitrogen Life Technologies, CA, USA). After 2 d, cells were harvested and analysed by flow cytometry. For LS174T cells, 1.5 x 10 6 cells were cultured overnight in a 6-well plate in Iscoves Modified Dulbeccos Medium (IMDM, Lonza, Basel, Switzerland) with 10% FCS and glutamine and 1.5 mL of virus was added with 1.5 uL of Polybrene (Santa Cruz Biotech, TX, USA). After 2 days, cells were harvested and analysed by flow cytometry.
In vivo bioluminescence imaging and acquisition of in vivo bioluminescence spectra.
'Animal work was carried out in accordance with Home Office regulations and guidelines as set out by the 1986 Animals Act (Scientific Procedures). Prior to imaging, mice were anaesthetised using 4% isoflurane and 2 L/min oxygen and during imaging were maintained using using 1.5% isoflurane and 1 L/ min oxygen and prior to imaging mice were ip injected with 2 mg of substrates. Mice were placed on a 37 o C heated bed in the Photon Imager Optima (Biospace Labs, Paris, France) and imaged for 20 min -40 min. In vivo bioluminescence spectra were collected by sequential 15 s, 30 s or 1 min acquisitions with the open filter, different filters and then open filter again. Spectra were corrected for signal decay during acquisition. Images were processed using M3 Vision software (Biospace Labs, Paris, France). Firefly luciferin potassium salt was purchased from Regis Technologies (IL, USA).   S284T with LH 2 (1) and iLH 2 (6).
Filter passband (+/-50nm) (nm) Normalised counts per minute/ cm 2 (cpm/cm 2 ) Bioluminescence spectra of native 1 (LH 2 ) and 6 (iLH 2 ). Bioluminescence spectra were acquired in a Photon Imager (Biospace Labs, Paris, France) which contains a sensitive intensified charge-coupled device (iCCD) detector with good red spectral sensitivity, by multiple consecutive 30s acquisitions using different band pass filters and correcting each output for overall signal decay. This enabled the determination of the proportion of light emitted at each wavelength for 1 (LH 2 ) and 6 (iLH 2 ) with different mutants.
Supplementary Figure S3. In vivo bioluminescence spectra of WT Fluc with LH 2 (1) and iLH 2 (6) esters in a subcutaneous model of colon carcinoma. bp filters with 50nm pass bands (mid-wavelength indicated in white) before imaging through the open filter. Mice were imaged for 30s or 1min with LH 2 and iLH 2 , respectively.
See Figure 4 legend (C) for details. Acquisition of spectra as in S4, but 15 s integration times used for both substrates.

General Experimental Details.
All manipulations were routinely carried out under an inert (Ar or N 2 ) atmosphere. All reagents were used as received unless stated. For the purposes of thin layer chromatography (tlc), Merck silica-aluminium plates were used, with uv light (254 nm) and potassium permanganate or anisaldehyde for visualisation. For column chromatography Merck Geduran ® Si 60 silica gel was used. Butyl lithium solutions were standardised with diphenyl acetic acid.