Patent Application: US-201414911821-A

Abstract:
the present invention relates to a genetically encoded fret - based biosensor to monitor the activity of matrix metalloproteinase 9 . mmp - 9 is an extracellular acting endopeptidase implicated in both physiological and pathological processes . a genetically encoded fret biosensor anchored in the cellular membrane allows studying the proteolytic activity of mmp - 9 with high spatiotemporal resolution at the exact region of mmp - 9 action on the cell . applicability of the biosensor , both in vitro and in vivo in living cells , has been demonstrated by ratiometric analysis of cleavage of the biosensor by a purified auto - activating mutant of mmp - 9 .

Description:
the genetically encoded fret - based mmp - 9 activity biosensor of the invention fills an important niche in the field of mmp - 9 detection . until now the mmp - 9 activity probes were created with clinical diagnosis in mind , which is understandable , given the role of mmp - 9 in the development of cancer . however , as recent years have shown , mmp - 9 plays a critical role in other processes , for example in the physiology of the brain . although probes , such as dq - gelatin , have been incredibly useful in studying that aspect of mmp - 9 role , they quite simply do not offer the required spatiotemporal resolution needed to answer numerous questions that have arisen . a genetically encoded , membrane - anchored fret - based mmp - 9 activity biosensor of the invention is better suited to elucidate the role of proteolytic activity of mmp - 9 in physiological and pathological processes . the biosensor of the invention utilizes a novel monomeric teal fluorescent protein ( mtfp1 ) that possesses superior spectral properties to cfp [ day , et al ., 2008 ]. it has previously been shown that mtfp1 forms a more efficient fret pair with the yellow fluorescent protein [ li and elledge , 2007 ]. however , other fluorescent proteins , such as clover fp [ lam et al ., 2012 ], can also be used in the biosensor of the invention . mtfp1 serves as a donor of energy while dual venus fluorescent proteins serve as energy acceptors . venus fluorescent protein , an improved variant of the yellow fluorescent protein , was selected for the fret acceptor . other fp can also be used as acceptor fp , for example mruby2 disclosed by lam et al ., supra . although the majority of fret - based sensors contain one donor protein and one acceptor protein , two acceptor fp proteins were introduced into the sensor to increase its fret efficiency . fret efficiency of a single donor and single acceptor system is defined as : where k t is the energy transfer rate , k r is the rate constant of all other deactivation processes and k f is the fluorescence decay rate . fret efficiency of a single donor and two acceptors system is given by the following equation : introduction of a second acceptor in a fret biosensor increases its fret efficiency , since the distance between fluorescent proteins in the biosensor of the invention was optimized to maximize the effective fret efficiency . since fret efficiency is determined not only by the distance between the donor and acceptor proteins , but also by the orientation , flexible linkers formed from several ggsggs or ggtggt repeats were employed and proved to be efficient in improving the fret efficiency . two glycine residues give the linker its flexibility , while a larger amino acid determines linear distance between proteins . the other factor determining the functionality of the biosensor of the invention is the mmp - 9 cleavage sequence . a screen of known mmp - 9 substrates has yielded neither a consensus sequence nor a secondary structure assumed by the mmp - 9 cleavage site . kridel et al . [ kridel , et al ., 2001 ] reported a family of short peptides cleavable by mmp - 9 using the phage display technology in an elisa format . thus the inventors have selected a consensus sequence n ′- prsls - c ′ suggested therein , which has been cloned into the biosensor of the invention . this sequence has previously been shown [ fudala et al ., 2011 ] as being indeed recognized by mmp - 9 . however , other amino acid sequences can also be used as mmp - 9 cleavage site ( as it has been discussed above ). as the proteolytic cleavage efficiency strongly depends on the accessibility of the cleavage site , which in turn is influenced by its secondary and tertiary structures , the inventors have decided to alter the structure of the linker positioned between mtfp1 and venus fluorescent protein in the biosensor of the invention , by placing the mmp - 9 cleavage site between two α - helices . the inventors have carried out the acceptor photobleaching ( ap ) experiments in order to rapidly screen generated biosensors and give a general indication of whether fret occurs . these experiments were never intended to provide a reliable quantification of the fret phenomenon . in - depth analysis of the fret properties of biosensors with the highest fret efficiency was performed with fluorescence lifetime imaging microscopy ( flim ). there is a slight discrepancy between fret efficiency values calculated from ap and flim experiments . however , the fret efficiency values based on ap are calculated for the entire cell ( therefore they include the cytoplasm , where fret is negligible due to the membranous localization of the biosensor ), whereas fret efficiency values based on flim measurements were calculated for much smaller regions , where fret was most pronounced . as the result of this analysis , it was found that the biosensor with α - helical linker has a higher fret efficiency than the biosensor with a loop - like linker . this difference is likely due to different higher order structures assumed by loop - like and α - helical linkers , with the latter one being more compact and thus bringing the donor and acceptors closer to each other . the mmp - 9 proteolytic activity leads to the release of dual venus proteins from the cell membrane and a decrease in the venus to mtfp1 fluorescence ratio . the biosensor is present predominantly in the cell membrane . the presence of a small fraction of the biosensor in the cytoplasm may indicate some level of sensor degradation . the biosensor is cleaved by mmp - 9 in an in vitro assay . the cleavage is not due to a spontaneous degradation of the protein and can be blocked by the addition of a broad spectrum mmp inhibitor . a baseline cleavage of the biosensor is observable in untreated lysate . since cell lysis was performed without protease inhibitors of any kind , the observed cleavage may be caused by endogenous mmp - 9 present in the hek293 cell lysate . the baseline cleavage can be , at least partially , blocked by matrix metalloproteinase inhibitors — both broad - spectrum ( gm6001 that blocks mmp - 1 , mmp - 2 , mmp - 3 , mmp - 8 and mmp - 9 ) and specific ( inhibitor i that selectively blocks mmp - 9 and mmp - 13 ). the fluorescence emission spectra collected from cell membranes of fixed hek293 cells expressing the biosensor and treated with the auto - activating mmp - 9 differ from those recorded from untreated cells . the contribution of mtfp1 to the fluorescence signal increases , implying the cleavage of the biosensor . the effect of mmp - 9 on the structure of biosensor has been followed with live cell imaging microscopy . this shows usefulness of the biosensor of the invention for investigation of the proteolytic activity of mmp - 9 in vitro , as well as in vivo in living cells . the genetically encoded fret - based mmp - 9 activity biosensor was assembled in the pdisplay plasmid ( clontech ). the mtfp1 gene was amplified from the pmtfp1 - n1 plasmid ( allele biotech ). the plasmid coding the venus gene was provided by jacek jaworski ( the international institute of cell biology , warsaw ). phusion hot start ii polymerase was purchased from thermo scientific ( formerly finnzymes ). xmai , sacii , nhel , aflii , agel , xbai , apal and bglii restriction enzymes were acquired in new england biolabs and thermo scientific ( formerly fermentas ). t4 dna polymerase required in the slic cloning was obtained from thermo scientific ( formerly fermentas ). dmem + glutamax ( high glucose 4 . 5 g / l ), fetal bovine serum and penicillin / streptomycin mix were purchased from sigma - aldrich . polyethylenoimine used for hek293 cell line transfection was acquired from fluka . proteoextract subcellular proteome kit was acquired from calbiochem and endofree plasmid dna maxi kit from qiagen . the α - gfp antibody was purchased from mbl (# 498 ), the α - myc antibody from santa cruz biotechnologies (# sc - 40 ), the α - n - cadherin antibody from bd biosciences (# 610920 ), the α - hsp90 antibody from stressgen (# sps - 771 ) and the α - histone h3 antibody from abcam (# ab10799 ). poly - l - lysine used to coat glass cover slips was purchased from sigma - aldrich . the auto - activating mmp - 9 was designed and purified as described previously [ michaluk , et al ., 2007 ]. the oligonucleotides were ordered either at sigma aldrich or genomed . the biosensor was cloned using the slic cloning methodology described in [ li , and elledge , 2007 ]. the fluorescent protein genes were amplified with the phusion hot start ii high fidelity polymerase ( thermo scientific ) using the following primers : the pdisplay plasmid was cleaved with xmal and sacll enzymes to generate single stranded ends . proper assembly of the venus - venus - mtfp1 tandem construct was confirmed using restriction enzyme analysis and sequencing . two restriction enzyme sites were introduced into the tandem construct : nhel site separated the venus 1 and venus2 fluorescent proteins , while aflii was cloned between the venus2 and mtfp1 genes ( sites marked in bold in primer sequences ). two oligonucleotides were cloned into these sites , each one coding a peptide linker designed to be flexible and provide spatial separation between the fluorescent proteins . oligonucleotides were composed of repeated segments separated with restriction enzyme cleavage sites ( agel and xbal ) to enable a rapid and simple adjustment of their length . an oligonucleotide ( coding linker ln1 ) with the following sequence was cloned into the aflii site : the agel restriction site is in bold font , the sequence coding the mmp - 9 cleavage site was underlined . the following oligonucleotide ( coding linker ln2 ) was cloned into the nhei site : the xbai restriction site is in bold . the biosensor constructed in the above fashion carried the mmp - 9 cleavage site within an unstructured loop , hence the name : biosensor with a loop - like linker . to construct a variant mmp - 9 activity biosensor with the mmp - 9 cleavage site located within α - helical structure , the linker between venus and mtfp1 in biosensor with a loop - like linker was cut out with the aflii enzyme and replaced with the following oligonucleotide ( seq no id : 9 ): eeeireafrvfprslslrhvmtnl ( seq id no : 10 ), where sequences given in bold represent α - helices . the membrane anchored mtfp1 was constructed in the pdisplay plasmid . the mtfp1 gene was amplified with the following primers — given in bold are restriction sites — apal in forward primer and bglii in reverse primer , respectively . the pcr product was cloned between these sites into the pdisplay plasmid . example 4 construction of mmp - 9 activity biosensors with varying linker lengths a series of variant biosensors with altered linker lengths were generated to perform fret efficiency optimization for the biosensor . the plasmid coding the biosensor with full length linkers was subjected to partial cleavage with either agel or xbal enzymes , religation and transformation into e . coli . restriction enzyme cleavage reactions were performed with increasing amounts of enzyme . the ln1 linker was cleaved for 2 h with an amount of agel sufficient to cut from 1 to 4 of its restriction sites — 0 . 85 u , 1 u , 1 . 5 u , 2 u , 2 . 5 u , 3 u , 3 . 5 u of agel were used . similarly the ln2 linker was cleaved with 6 u , 8 u , 10 u , 12 u , 14 u , 16 u 18 u or 20 u for 2 h , to cleave the plasmid in 1 to 4 xbal sites . clones were analyzed using pcr to determine the linker length within the biosensors and sequenced . the following biosensors were received : 1 - 1 , 2 - 1 , 3 - 1 , 4 - 1 , 5 - 1 , 6 - 1 , 7 - 1 , 8 - 1 , 1 - 2 , 2 - 2 , 8 - 2 , 1 - 3 , 2 - 3 , 3 - 3 , 4 - 3 , 5 - 3 , 6 - 3 , 7 - 3 , 8 - 3 , 1 - 4 , 2 - 4 , 8 - 4 , 1 - 5 , 2 - 5 , 8 - 5 , 1 - 6 , 2 - 6 , 8 - 6 , 1 - 7 , 8 - 7 , 1 - 8 , 2 - 8 , 3 - 8 , 4 - 8 , 5 - 8 , 6 - 8 , 7 - 8 , 8 - 8 , wherein the first digit in the number pair designating the biosensor variant indicates a number of ggsggr hexapeptide repeats in ln2 linker and the second digit in the number pairs indicates a number of ggtggt hexapeptide repeats in ln1 linker . routine fret optimization and testing of the sensor were performed in the hek293 cell line . cells were cultured in dmem ( 4 . 5 g / l glucose )+ 10 % fbs + 1 % p / s in 37 ° c ., 5 % co 2 . plasmids coding the sensors ( identified in the proceeding examples ) were purified with the qiagen endo free plasmid maxi kit . dna to be transfected was mixed with pure dmem and polyethylenoimine ( pei ) ( 5 μg / μl ), left for 10 minutes at room temperature , and then transferred to cell culture . cells were incubated with dna - pei complexes for 4 h , then the medium was replaced with a fresh one . cells intended to be imaged on confocal microscope were cultured on glass cover slips coated with poly - l - lysine . example 6 ap / flim analysis of the cells expressing mmp - 9 activity biosensors two days post transfection the cells obtained in example 5 were fixed with 4 % pfa , 3 % sucrose in pbs and microscope slides were prepared . acceptor photobleaching ( ap ) experiments were performed on leica sp5 microscope with 63 × na ( 1 . 4 ) oil immersion objective . images were acquired at 1024 × 1024 pixels . mtfp1 was imaged with the 458 nm line of an argon laser set to 20 %. fret efficiency of the sensors was determined by ap of the venus with high power 514 nm laser and measuring the increase in the intensity of the fluorescence of mtfp1 . sensors determined to have highest fret efficiency were further analyzed with the fluorescence lifetime imaging microscopy ( flim ) on leica sp2 microscope . the apparent fret efficiency value of variant sensors from ap data was calculated using the following equation : where f d is the fraction of donor participating in the fret complex , f da and f d are the background subtracted and acquisition bleaching corrected pre - and post - bleach mtfp1 fluorescence intensities , respectively . the acquisition bleaching corrected post - bleach mtfp1 intensities were calculated as where f d b and f d r refer to mtfp1 intensities of the bleach and reference region of interest , and pre and post refer to pre - bleach and post - bleach measurements . fret efficiency values from flim data were calculated with the following equation where τ d is the lifetime of the donor in the absence of the acceptor ( in our case the membrane anchored mtfp1 ) and τ da is the lifetime of the fret - based mmp - 9 activity biosensor and a da and a d represent the amplitude of individual decay components [ zeug , et al ., 2012 ]. error values were estimated using the gaussian noise propagation equation example 7 fluorescence emission spectra collection for cells transfected with mmp - 9 activity biosensors lambda stack acquisition was performed on zeiss lsm780 microscope equipped with 63 × na ( 1 . 4 ) oil - immersion objective at 1024 × 1024 pixels . the 458 nm line of an argon laser was used for excitation and 32 lambda channels were acquired , at 9 nm steps . acquired lambda stacks were analysed with fiji imagej software by measuring the average brightness of the plasma membrane in each channel . recovered sensor spectra were normalized by having the area under the spectrum plot equaling 1 . cell fractioning experiments were performed using the calbiochem proteoextract subcellular proteome extraction kit . sensor was detected on western blot using the anti - myc antibody . quality of the cell fractioning was tested on western blot with the following antibodies : anti - hsp90 , anti - n - cadherin and anti - histone h3 . two days post - transfection hek293 cells were washed one with pbs , scraped from the plate and lysed for 1 h at 4 ° c . with the following buffer : 50 mm tris - ci ph 7 . 5 , 1 % triton x - 100 , 10 mm cacl 2 , 0 . 02 % nan 3 , 1 μm zncl 2 . the lysis was performed without protease inhibitors since it was feared that they might block the activity of our auto - activating mmp - 9 . the lysate was then centrifuged at 13400 rpm for 15 ′ at 4 ° c . to remove cell debris . equal amount of the cleared lysate were used in the subsequent reactions . either 400 ng ( final concentration 10 μg / ml ), 1 . 2 μg ( final concentration 30 μg / ml ) of auto - activating mmp - 9 or 400 ng ( final concentration 10 μg / ml ) of inactive mmp - 9 were added to the reactions . gm6001 inhibitor was used in 25 μm final concentration . reactions were stopped at either 30 ′, 1 h , 4 h or after overnight incubation at 37 ° c . with the addition of sds - page sample buffer and heating to 100 ° c . for 10 minutes . sensor was detected on western blot with the anti - gfp antibody . example 10 cleavage of the mmp - 9 activity biosensors in the cell culture two days post - transfection with the mmp - 9 activity biosensors , the culture medium was replaced with pure dmem . cells were incubated for 30 min . with 400 ng of auto - activating mmp - 9 ( final concentration — 800 ng / ml ) and fixed with 4 % pfa , 3 % sucrose in pbs . lambda stacks were acquired as previously described . example 12 live imaging — ratiometric analysis of cells transfected with mmp - 9 activity bio sensors the hek293 cell line was cultured on glass bottom microwell dishes ( mattek corporation ). the cells were transfected with a plasmid coding the biosensor with α - helical liner . two days post - transfection the cells were transferred to a zeiss lsm780 microscope fitted with incubator and imaged using a water - immersion 40 × objective . a single optical slice of the cells was captured at the 1024 × 1024 pixel resolution every 30 s with linear unmixing of the donor and acceptor fluorescence spectra performed in real time . acquisition was performed for 30 min . 5 min after the start of image acquisition the cells were either mock treated with pure dmem , auto - activating mmp - 9 diluted in dmem ( final concentration — 460 ng / ml ) or inactive mmp - 9 similarly diluted in dmem to the same final concentration . data analysis was performed in the custom written software under matlab suite . the venus / mtfp1 ratio was calculated for each pixel and plotted against the time elapsed from the start of the experiment . the introduction of linkers with adjustable lengths allowed a rapid formation of 38 biosensor variants . biosensors of the invention with the highest fret efficiency were identified with the ap technique ( fig1 b ) and analyzed using flim to confirm ap - based fret efficiency calculations . table 1 presents fret efficiency values calculated from flim data for the biosensors variants with the highest fret efficiencies in ap experiments . error values were estimated using the gaussian noise propagation equation . the naming scheme of the variants is as follows : x - y , where x — a number of hexapeptide repeats between both venus fp ( linker ln2 ), y — a number of hexapeptide repeats between venus fp and mtfp1 ( linker ln1 ). a biosensor ( named 7 - 1 in table 1 ) with a long linker between the two venus fp ( seven repeats ggtggttctggttctaga ( seq id no : 13 ) in its dna sequence ) and a short linker between the second venus and mtfp1 ( one ggaggaaccggtggaact repeat ( seq id no : 14 ) in its dna sequence ) has the highest fret efficiency of all obtained biosensor loop - like linker variants and the fret efficiency was found to be e = 0 . 20 ± 0 . 03 ( standard deviation value )— fig2 a . the biosensor with α - helical linker has a higher acquisition - bleaching corrected effective fret efficiency 0 . 26 ± 0 . 02 in comparison to the biosensor with the loop - like linker — fig2 b . given the higher fret efficiency exhibited by the biosensor with α - helical linker , it was used in further studies . the mmp - 9 activity biosensor of the invention localizes at the cellular membrane . this was confirmed by direct visualization of the biosensor in living hek293 cells ( fig1 c ) and through cell fractionation followed by western blot analysis of collected fractions ( fig1 d ). fractionation confirms that the vast majority ( 87 %± 6 %) of the biosensor localizes in membranes with a small percentage in the cytoplasm . no biosensor was observed in the nucleus . control western blots confirmed the purity of collected fractions . the mmp - 9 activity biosensor is cleaved in vitro by a human auto - activating mmp - 9 ( fig3 ). significantly more enzyme was used ( standard concentration of auto - activating mmp - 9 in experiments is 400 ng / ml [ michaluk et al ., 2009 ]) in these reactions to make sure that the entire biosensor pool was cleaved . the biosensor is already partially cleaved ( 14 . 2 ± 0 . 4 %— see fig3 b , control lanes ; value normalized to full length biosensor intensity in control lanes ) in transfected but otherwise untreated hek293 cell line , which can be readily seen on the western blot ( fig3 ). the in vitro cleavage of the biosensor with α - helical linker is not due to spontaneous degradation of the protein , though a slight increase in the amount of a cleaved form of the biosensor is observed over time ( fig3 ). inactive human mmp - 9 does not cleave the biosensor ( fig3 ). the cleavage can be blocked by the addition of a broad spectrum gm6001 matrix metalloproteinase inhibitor to the final concentration of 25 μm ( fig3 ). iv . biosensor cleavage in the hek293 cell culture — fluorescence emission spectra of fixed cells analysis of the fluorescence emission spectra collected from hek293 cells incubated with the auto - 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