WO2021158184A1 - Method of producing modified amyloid precursor protein anchored with detectable moieties and modified amyloid peptides derived thereof - Google Patents

Abstract

The present disclosure relates to a method of producing a modified amyloid precursor protein capable of emitting self-reporting signal. The method generally comprises the steps of providing polynucleotide sequence SEQ ID NO. 1 encoding for an amyloid precursor protein (APP); modifying SEQ ID NO. 1 through genetic code expansion to replace a natural amino acid encoded at a predete1mined position within a region encoding for an amyloid β peptide in the SEQ ID NO. 1 with an unnatural amino acid; expressing the modified SEQ ID NO. 1 to obtain the APP carrying the amyloid β peptide with the unnatural amino acid; and anchoring a fluorophore to the unnatural amino acid carried on the APP to acquire the modified APP.

Description

METHOD OF PRODUCING MODIFIED AMYLOID PRECURSOR PROTEIN ANCHORED WITH DETECTABLE MOIETIES AND MODIFIED AMYLOID PEPTIDES DERIVED THEREOF

Technical Field

The present disclosure relates to a method of producing modified or synthetic amyloid precursor protein (APP) equipped with at least one signaling probe or detectable moiety, which is strategically placed to enable at least a given region or segment attached with the signaling probe, which becomes detectable corresponding to one or more bioprocesses occurred in vivo. Also, the present disclosure includes a modified or synthetic APP generated from the mentioned method and technical applications of produced modified APP.

Background

Amyloid β (Ab) peptides are 36-43-amino acid peptide species generated from sequential proteolytic processing steps of APP, notably by the b-site APP cleaving enzyme (BACE) then by g-secretase¹. Deposition ofAβ plaques is the hallmark feature of Alzheimer’s disease² thus reasons and processes relating to the formation of theAβ peptides under various circumstances have attracted numerous research attentions. For instance, United States patent no. 6277826 discloses a protein-based compound claimed to be an effective modulator for aggregation of Ab peptides through its binding affinity towards the peptides. Other the modulators ofAβ peptides can be found in WO 1996028471, US6689752, US7345022, etc. WhileAβ peptides’ in vitro aggregation kinetics³ as well as individual processing enzymes involved in the generation of the peptides are relatively well understood, much less is known about the cellular dynamics of processing of APP to generateAβ peptides and their aggregation — due to the lack of tools to visualize newly and natively/freshly processedAβ peptides in cells. For example, Dale et al. offers a method for detectingAβ peptides solubilized in body fluid by binding specific epitope ofAβ peptides to a binding substance as setting out in United States patent no. 5593846. Similarly, United States patent application no. 201600771 1 describes another method for detecting presence of misfoldedAβ peptides solubilized in a sample through reacting the sample with monomeric foldedAβ peptides. There are no doubts about competence of these methods in yielding the desired results, but they lack the capacity to better illustrate formation and aggregation ofAβ peptides within cells in a substantially or almost real-time manner. Furthermore, traditional protein tagging techniques such as GFP fusion produceAβ with aberrant oligomerization kinetics, presumably due to interference from GFP size as well as its tendency to oligomerize⁴. Moreover, simple tagging of Aβ with genetic tags, epitope tags or FlAsFI⁵ cannot recapitulate the cellular microenvironments in the endocytic, retrograde, and secretory pathways — whereAβ can be generated from APP⁶ — that likely influence Ab modifications, aggregation, transport, and interactions with other biomolecules and organelles in the cell⁷. Understanding the spatiotemporal context in which Aβ is produced and accumulated may provide insight into pathogenicity ofAβ as well as therapeutic development targeted toward APP andAβ for Alzheimer’s disease. In view of that, there presents the need of an improved instrument and its production to aid one for visualizing, studying and/or analysing formation of Ab peptides subjecting to different reactions in vivo, preferably in a real-time fashion.

Summary

One object of the present disclosure is directed to provide a method of producing a modified APP. The method allows the modified APP produced with the capability to constantly emit a detectable signal within the cells which the modified APP will be subjected to various catalytic reactions in yielding different compound includingAβ peptides.

Another object of the present disclosure is to disclose a method of producing a modified APP that the method enhances detectability or visualization of the modified APP within the cells in real-time by anchoring one or more detectable moieties. More specifically, the modified APPs acquired through some embodiments of the disclosed method are able to release two or more distinctive signals for identification of two or more different compounds derived from the modified APP.

Further object of the present disclosure relates to a modified APP or amyloid peptide, which is an effective instrument to assist one in observing, detecting, studying and/or analyzing the Ab peptides newly formed in vivo from the modified APP such that better understanding and treatment of Alzheimer’s disease can be developed.

According to several embodiments of the present disclosure, a method of producing a modified amyloid precursor protein capable of emitting self-reporting signal is disclosed. The method generally comprises the steps of providing polynucleotide template having a sequence SEQ ID NO. 1 encoding for an amyloid precursor protein (APP); modifying the polynucleotide template through genetic code expansion to replace a natural amino acid encoded at a predetermined position within a region encoding for an amyloid β peptide in the SEQ ID NO. 1 with an unnatural amino acid; expressing the modified polynucleotide template to obtain the APP carrying the amyloid β peptide with the unnatural amino acid; and anchoring a fluorophore to the unnatural amino acid carried on the APP to acquire the modified APP.

For several embodiments, the predetermined position is any one of H609, H610 and Q611. The unnatural amino acid incorporated at one of these positions has no or almost no substantial effect on the maturation and/or trafficking of the modified APPs generated in vivo from the polynucleotides template SEQ ID NO. 1.

For more embodiments, the method further comprises attaching fluorescent protein to a C- terminus of the modified APP.

In more embodiments, the method includes attaching a protein-based labelling tag to a C- terminus of the modified APP and carrying a quencher on the protein-based labelling tag, wherein the quencher is fashioned to absorb or nullity signal emitted from the fluorophore such that the signal emitted by the fluorophore only becomes detachable upon disassociating the amyloid β peptide from the modified APP.

In some embodiments, the unnatural amino acid is bicyclononyne-lysine, cyclopropane- lysine, alkyne-lysine, trans-cyclooctene-lysine, any derivatizable amino acid, or any fluorescent amino acid.

Another aspect of the present disclosure refers to a modified amyloid precursor protein or amyloid peptide. The modified amyloid peptide essentially comprises an extracellular domain; a transmembrane domain; a cytoplasmic domain having a C-terminus being further extended from the transmembrane domain; an amyloid β peptide segment interposing in between the extracellular domain and the transmembrane domain, the amyloid β peptide segment comprising an unnatural amino acid anchored with a fluorophore capable of constantly emitting a detectable signal. Preferably, the unnatural amino acid is introduced by replacing a natural amino acid found at position H609, H610 or Q611 as setting forth in SEQ ID NO. 2

For a number of embodiments, the modified amyloid precursor protein or amyloid peptide may further comprise a fluorescent protein attached to the C-terminus in addition to the fluorophore.

Brief Description of Drawings

Figure 1 shows representative schematic embodiments of the disclosed modified APP with reporting capability, where (a) is the modified APP produced with fluorophore and fluorescent protein respectively appended on the amyloid β peptide segment and C-terminus and (b) is the modified APP produced with fluorophore and fluorescent quencher respectively appended on the amyloid β peptide segment and C-terminus;

Figure 2 shows (a) scheme for site selection of APP695 for mutation to TAG, (b) Western blot analysis of lysates (20 pg) of FTEK293 cells transfected with various APP(TAG)-α-myc mutant plasmids incubated 45 hours in the presence of 60 μM BCNK and analysed using anti-α-myc antibody with TAG mutation being represented in * whereas APPm and APPim indicate the bands corresponding to mature- APP and immature- APP, (c) Western blot analysis of lysates (20 pg) of HEK293 cells transfected with various APP(TAG)-α-myc mutant plasmids incubated 45 hours in the absence of 60 pM BCNK and analysed using anti-α-myc antibody with TAG mutation being represented in * whereas APPm and APPim indicate the bands corresponding to mature- APP and immature- APPrespectively; and (d) graph about quantification of intensity ratio between mature-APP and immature- AP;

Figure 3 shows (a) analysis results of HEK293 cells transfected with APP(TAG)-Myc mutant plasmids, PylRS-AF and Pyl-tRN2_CUA then incubated with 60 pM BCNK for 45 hours that the APP (BCNK) were labelled in vivo by 1.2 μM tetrazine-cy5 (tetrazine-cy5 channel, pink colour) prior to post fixation and analyzed by anti-α myc antibody ((α-myc/Alexa488) channel, green colour) while the expression of aminoacyl-tRNA synthetase was analysed by anti-FLAG antibody ((α-FLAG/Alexa568) channel, red colour), (b) graph about quantification of intensity mean value of α-myc/Alexa488 for expressed APPs modified at several predetermined sites, (c) graph about quantification of intensity mean value of tetrazine-cy5 for expressed APPs modified at several predetermined sites, (d) graph about quantification of intensity mean value and intensity ratio between tetrazine-cy5 and α-myc/Alexa488 for expressed APPs modified at several predetermined sites (values shown are mean ± SEM. Each condition represents over 50 cells analyzed);

Figure 4 is a gel picture showing Western blot analysis for APP(E1609TAG)-EGFP expression that lysates (20 μg) of HEK293 cells transfected with APP(H609TAG)-EGFP were incubated 45 hours in presence (+BCNK) and absence (-BCNK) of 60 μM BCNK;

Figure 5 shows fluorescence images of de novo generated Aβ reporter where HEK293 cells transfected with APP(H609TAG)-EGFP plasmid, PylRS-AF and Pyl-tRNA_CUA were incubated with 60 μM BCNK for 45 hours followed by labelling the protein in vivo by 1.2 μM tetrazine-cy5 (cy5 channel, pink colour) at 4 °C for 30 minutes (indicated as 0 hour thereof) and continuously grown for another 2 hours for APP processing, post fixation and analyzed by confocal electronic microscope with the expression of APP(H609TAG)-EGFP being shown in green colour via EGFP;

Figure 6 shows fluorescence images of exogenously expressedAβ peptides acquired from an expression plasmid without subjecting the expressedAβ peptides for in vivo processing compared to the results of freshly or natively processedAβ peptides with HEK293 cells transfected with rRB-HA-Ab40 or rRB-HA-Ab42 plasmid (expression under EFla promoter) being cultured for 45 hours followed by fixed and immunofluorescently labelled with anti -HA antibody and a secondary antibody conjugated to Alexa Flour 568; and

Figure 7 shows fluorescence images of localization of APP andAβ to the trans-golgi network (TGN). HEK293 cells were transfected with a plasmid expressing an organelle marker mApple- TGN38 (TGN marker) or mApple-Rab7a (late endosome marker), along with other genetic code expansion transgenes where cells were labeled with cy5-tetrazine and incubated for 2 hour post- labeling at 37 °C;

Figure 8 shows western blot picture where HEK293 cells transfected with transgenes encoding APP(H609TAG)-HaloTag followed by incubation in the presence and absence of 60 μM BCNK for 45 hours the cell lysates were analyzed with western blot using anti-myc antibody; Figure 9 shows fluorescence images about dose-dependent quenching of cy5 fluorescence on APP with QSY21-C1. HEK293 cells were transfected with transgenes expressing APP(H609TAG)-HaloTag, PylRS-AF and Pyl-tRNACUA and incubated with 60 μM BCNK for 45 hours, where cells were labeled live with 1.2 μM tetrazine-cy5 and indicated concentrations of QSY21-C1 at 4 °C for 30 minutes, immediately fixed, and then immunostained the APP- FlaloTag via its C-terminal myc tag for imaging;

Figure 10 shows zoom-ins of cell samples prepared as in Figure 9, which were further incubated for 2 hours at 37 °C post-labeling to permit APP internalization and processing;

Figure 11 is a gel picture showing that the peptides generated from the reporter having peptide sizes consistent with amyloid-b peptide species in an experiment where HEK293 cells transfected with transgenes encoding APP(H609TAG), APP(H609TAG)-EGFP, or APP(H609TAG)-FialoTag were incubated with 60 μM BCNK for 45 hours, labeled with 1.2 μM tetrazine-cy5, lyzed immediately after labeling or further incubated for 2 hours at 37 °C post- labeling before cell lysis, and analyzed by in-gel fluorescence with cy5 laser excitation and filter settings; and

Figure 12 shows polynucleotide and peptide sequence respectively setting forth in SEQ ID NO. 1 and SEQ ID NO. 2.

Detailed Description

For the purpose of facilitating an understanding of the disclosure, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the disclosure, its construction and operation and many of its advantages would be readily understood and appreciated.

As used herein, the phrase “in embodiments” means in some embodiments but not necessarily in all embodiments.

As used herein, the terms “approximately” or "about", in the context of concentrations of components, conditions, other measurement values, etc., means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value, or +/- 0% of the stated value.

The term “polypeptides” used herein throughout the disclosure refers to a chain of amino acids linked together by peptide bonds but with a lower molecular weight than protein. Polypeptides can be obtained by synthesis or hydrolysis of proteins. Few polypeptides can be joined together by any known method in the art to form a functional unit.

The term “detectable moieties” or “reporting moieties” used herein throughout the detail description refers to compounds which capable of releasing signal that is detectable either by a device or naked eye to reveal the presence of the formed of a complex and the like such as antigen-antibody complexes to ascertain the diseased state of the test subject. The signal can be in the form of the low frequency wave or color changes due to substrate-enzyme activity as in the ELISA or any other modified methods derive thereof.

The term “quencher” as used herein refers to a moiety that absorbs at least some of the intensity of a fluorescent emission. Quenchers can be categorized as fluorescent quenchers and dark quenchers (also referred to as non-fluorescent quenchers).

According to one aspect of the present disclosure, a method of producing a modified amyloid precursor protein capable of emitting self-reporting signal is described hereinafter. Preferably, the method comprises the steps of providing polynucleotide template having sequence SEQ ID NO. 1 encoding for an amyloid precursor protein (APP); modifying the polynucleotide template of SEQ ID NO. 1 through genetic code expansion to replace a natural amino acid encoded at a predetermined position within a region encoding for an amyloid β peptide in the SEQ ID NO. 1 with an unnatural amino acid; expressing the modified SEQ ID NO.1 to obtain the APP carrying the amyloid β peptide with the unnatural amino acid; and anchoring a fluorophore to the unnatural amino acid carried on the APP to acquire the modified APP. It is important to note that the polynucleotide template may set out other variants of APP rather than mere sequence of SEQ ID NO. 1 for other embodiments of the disclosed method such that observation and analytic works can be performed in relation to these variants of APP. The codon encoding for the unnatural amino acid of the disclosed method is incorporated to the polynucleotide template through genetic code expansion. Through the genetic code expansion, the codon of the unnatural and non-standard amino acid can be artificially added into the polynucleotide template for subsequent expression later. More preferably, the codon encoding for the unnatural amino acid is recognizable by tRNA and/or tRNA synthase when the modified APP is expressed in the host cells. There are a number of unnatural or non-standard amino acids such as bicyclononyne-lysine, cyclopropene-lysine, alkyne-lysine, trans-cyclooctene-lysine, any derivatizable amino acid, or any fluorescent amino acid known in the field that suitable for the application of the present disclosure. With reference to the more preferable embodiments, bicyclononyne-lysine amino acid, but not limited to, is employed for the disclosed method.

As described in the setting forth, some embodiments of the disclosed method replace natural amino acid encoded within the polynucleotide temple at one of the predetermined positions. Preferably, the predetermined position is any one of H609, H610 and Q611 as of the polypeptide template SEQ ID NO. 2, which is the preferred variant of APP used in the present disclosure as encoded by SEQ ID NO. 1. It was found by the inventors of the present disclosure that these predetermined positions are free from subjecting to post-translational modification in cells thus rendering the intended modification being retained for succeeding treatment to anchor the detectable moieties and/or observation about metabolism of the expressed modified APPs in vivo. Moreover, the present disclosure discovered that the modifications made at one of these predetermined sites have no substantial or almost no substantial negative impact on the metabolism of the expressed modified APPs in cells. With the mentioned modifications, one can truly visualize, observe, analyze, and/or study metabolism of the modified APPs freshly expressed or introduced into the host cells. Also, these predetermined positions are preferably resided within the segment encoding for theAβ peptides, which will be cleaved off upon metabolization of the modified APPs within the cells. Particularly, the modifications at these predetermined positions allow one or more detectable moieties such as fluorophore to be anchored onto the separableAβ peptides to better illustrate metabolism of the modified APPs.

In a number of embodiments, the cells or cell lines which are applicable in the present method for expressing the modified APPs or amyloid peptides are HEK293T, HepG2, neuronal cell lines, primary neurons, etc. The modified APPs or amyloid peptides can be yielded biologically using one of the known compatible cell types through any known approach in the field without departing from the scope of the disclosed method. After the expression, the derived modified APPs or amyloid peptides further undergo derivatization chemistry, such as via inverse electron- demand Diels-Alder reaction, to finally attach the fluorophores onto the modified APPs or amyloid peptides. The fluorophore has fluorescent spectra ranging from 350-750 nm, such as Cy2, Cy3, Cy5 or Cy7, which is able to consistently emit a first signal detectable by the compatible tools.

For more embodiments, the disclosed method comprises attaching fluorescent protein to a C- terminus of the modified APP. The fluorescent protein is configured to release a readable signal, namely a second signal, being distinctive from the first signal originated from the fluorophores. The two variants of the detectable or reporting moieties allow real time illustration of intracellular APP processing via the fluorescent signals, the first and second signals respectively derived from theAβ segment and the C-terminus of APP. The illustration may indicate the modified APPs have been fully metabolized or processed in the cells with theAβ peptide segment being cleaved away when only a single signal, particularly the second signal, is detected. The two distinctive signals, the first and second signals, facilitates formation and separation of theAβ peptides from the modified APPs. Preferably, the fluorescent protein is cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), enhanced orange fluorescent protein (OFP), enhanced green fluorescent protein (eGFP), modified green fluorescent protein (emGFP), enhanced yellow fluorescent protein (eYFP) and/or monomeric red fluorescent protein (mRF^’P) and/or modified derivatives.

Pursuant to more preferred embodiments, the method may further comprise attaching a protein- based labelling tag to a C-terminus of the modified APP and carrying a quencher on the protein- based labelling tag as shown in Figure lb. Specifically, the quencher is fashioned to absorb, dampen or nullify signal emitted from the fluorophore such that the signal emitted by the fluorophore only becomes detachable upon disassociating or separating the amyloid β peptide from the modified APP. The protein-based labelling tag can be BCCP (Biotin Carboxyl Carrier Protein), Glutathione-S-transferase-tag, Halo-tag, Maltose binding protein-tag, Nus-tag, Thioredoxin-tag, Fc-tag, SNAP-Tag, CLIP-Tag, or any other protein or peptide-based labelling tag. Preferably, Halo-tag is used in the examples shown hereinafter.

Another major aspect of the present disclosure refers to a modified amyloid precursor protein or amyloid peptide, which is applicable for illustrating the processing, metabolism and/or formation of freshAβ peptide in vivo. The disclosed modified APP or amyloid peptide preferably comprises an extracellular domain; a transmembrane domain; a cytoplasmic domain having a C- terminus being further extended from the transmembrane domain; and an amyloid β peptide segment interposing in between the extracellular domain and the transmembrane domain, the amyloid β peptide segment comprising an unnatural amino acid anchored with a fluorophore capable of constantly emitting a detectable signal. It is crucial to note that the amino acid sequence of the produced APPs or amyloid peptides has been substantially set out in SEQ ID NO. 2, except the various modifications potentially introducing the unnatural amino acid. The different domains and segments of the disclosed modified APPs or amyloid peptides is preferably arranged in tandem as described in the foregoing, but these domains and segments may eventually fold into and adapt a 3D conformational structure similar of those depicted in Figure 1 a and 1 b. In accordance with the preferred embodiments, the unnatural amino acid of the disclosed modified APPs or amyloid peptides is introduced by way of replacing a natural amino acid found at position H609, H610 or Q611 as setting forth in the polypeptide template of SEQ ID NO. 2. Particularly, the codon encoding for the unnatural amino acid is incorporated to the polynucleotide template SEQ ID NO. 1 through genetic code expansion as setting forth in the abovementioned methods. By the genetic code expansion, the codon of the unnatural and non-standard amino acid can be artificially added into the polynucleotide template then further expressed and chemically activated to produce the modified APPs or amyloid peptides. The codon encoding for the unnatural amino acid shall be recognizable by tRNA and/or tRNA synthase for expression of the APPs or amyloid peptides in the host cells. Preferably, the unnatural or non-standard amino acids employed in the present disclosure is any one of bicyclononyne-lysine, cyclopropene-lysine, alkyne-lysine, trans-cyclooctene-lysine, any derivatizable amino acid, or any fluorescent amino acid. After the expression, the derived APPs or amyloid peptides is subjected to cycloaddition via inverse electron-demand Diels-Alder reaction for anchoring the fluorophores to yield the desired modified APPs or amyloid peptides. As regards to the fluorophores usable in the present disclosure, they can of those conventionally used in the art including but not limited to xanthene moieties; coumarin moieties and cyanine moieties. The examples of xanthane moieties are fluorescein derivatives and rhodamine derivatives. For coumarin moieties, they can be hydroxycoumarin, methylcoumarin and aminocoumarin. Representative examples of cyanine moieties can be Cy2, Cy3, Cy5 or Cy7. Moreover, dyes such as Alexa dyes, cyanine dyes, Atto Tec dyes, and rhodamine dyes can be used in some embodiments of the present disclosure as well. Preferably, the fluorophore is any one of Cy2, Cy3, Cy5 and Cy7, which is able to consistently emit a first signal detectable by the compatible tools.

As shown in the embodiments of Figure la, the modified APP or amyloid peptide may comprise a fluorescent protein attached to the C-terminus. Preferably, the fluorescent protein is configured to release a readable signal, namely a second signal, being distinctive from the first signal released from the fluorophores. The two variants of the detectable or reporting moieties featured in the disclosed modified APPs or amyloid peptides allow real time illustration of intracellular APP processing via the fluorescent signals, the first and second signals respectively derived from theAβ segment and the C-terminus of APP or amyloid peptide. Preferably, the fluorescent protein is cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), enhanced orange fluorescent protein (OFP), enhanced green fluorescent protein (eGFP), modified green fluorescent protein (emGFP), enhanced yellow fluorescent protein (eYFP) and/or monomeric red fluorescent protein (mRFP) or modified derivatives.

Further embodiments of the disclosed modified APPs or amyloid peptides may include a protein- based labelling tag attached to the C-terminus as well. This protein-based labelling tag can be any of BCCP (Biotin Carboxyl Carrier Protein), Glutathione-S-transferase-tag, Halo-tag, Maltose binding protein-tag, Nus-tag, Thioredoxin-tag, Fc-tag, SNAP-Tag, CLIP-Tag, or any other protein or peptide-based labelling tag. The protein-based labelling tag serves as a platform for further attachment of a quencher. The quencher being anchored to the protein-based labelling tag is fashioned to absorb or nullify the signal emitted from the fluorophore when both fluorescent quencher and fluorophore are appended to the same compound, the modified APP or amyloid peptidesin this situation. Such arrangement ensures the signal emitted by the fluorophore only becomes detachable upon disassociating or cleaving the amyloid β peptide from the modified APP or amyloid peptide in the cells.

Accordingly, the quencher employed in the present disclosure can be a fluorescent quencher. The fluorescent quencher is a moiety, typically a fluorophore, that can absorb the fluorescent signal emitted from a source of fluorescence at a first wavelength, for example but not limited to, a nucleic acid dye associated with a double-stranded segment of nucleic acid, and after absorbing enough fluorescent energy, the fluorescent quencher can emit fluorescence at a second wavelength that is characteristic of the quencher, a process termed “fluorescent resonance energy transfer” or FRET. In some embodiments, the fluorescent quencher can be any of QSY21, Dabcyl, Malachite green and TAMRA. For more preferred embodiments, the non-fluorescent quenchers such as Dabcyl, BHQ-1, BHQ-2, BHQ-3, Iowa Black, QSY series quencher such as QSY 7 and QSY 21, Absolute Quencher; or Eclipse non-fluorescent quencher are used. The manner which the quencher and the protein-based labelling tag are joined can be of any approach known in the field. Likewise, lodgment of the protein-based labelling tag can be performed using known method without departing from the scope of the present disclosure.

The following example is intended to further illustrate the disclosure, without any intent for the disclosure to be limited to the specific embodiments described therein.

Example 1

To label and image natively processed Ab, the present disclosure first adapted genetic code expansion for labelling theAβ segment of amyloid precursor protein (APP). Genetic code expansion uses an orthogonal aminoacyl-tRNA synthetase/tRNA pair to direct incorporation of a designer unnatural amino acid in place of a natural amino acid, at any user-defined or predetermined site, via the use of an amber stop codon (UAG)⁸. In more specific, The APP mutants were produced by site-directed mutagenesis method using pGFP-n1-APP plasmid (Addgene #69924) as a template. The site-directed mutagenesis was carried out by PCR amplification using mutagenic primers to create the amber codon (TAG (*)) at the desired position of the amyloid-b (Ab) segment of the APP including H602*, D603*, H609*, H610* and Q611 *. Subsequently, all mutants as well as APP-wild type in pGFP-n1-APP plasmid were cloned into previously reported plasmid (pPB) for genetic code expansion in mammalian cells using restriction cloning (NheI and Notl restriction site (NEB). For construction of APP reporter plasmid, pGFP-n1-APP(H609TAG) was digested with Nhe\ and Notl restriction enzymes and sub-cloned into pPB vector to generate pPB_APP(H609TAG) _4xPyIT.To incorporate a fluorophore at a defined site within Ab, the present disclosure used a two-step labelling strategy: the unnatural amino acid bearing a biorthogonal handle is first incorporated co-translationally into proteins via genetic code expansion; then, the functional handle is further derivatized via biorthogonal chemistry. The unnatural amino acid of choice is bicyclononyne-lysine amino acid⁹, which can be incorporated efficiently into proteins via an engineered pyrroylysyl-tRNA synthetase/tRNA pair¹⁰ and then further derivatized with an inverse-electron-demand Diels- Alder cycloaddition with tetrazine-fluorophores¹¹.

The present disclosure selected several amino acid positions within theAβ portion of APP695 (the predominant neuronal isoform of APP¹² that are not involved in post-translational modifications as illustrated in Figure 2a, to be replaced with BCNK. Among the positions tested, three amber variants of APP — H609TAG, H610TAG, and Q611TAG — showed strong BCNK- dependent expression to produce full-length APP (Figure 2b and 2c). These APP(BCNK) variants all produced two major bands corresponding to mature APP and immature, core- glycosylated APP¹³ ), with mature: immature APP intensity ratios being similar to that of the wild-type APP construct (Figure 2d), suggesting that BCNK incorporation into APP does not affect its maturation and trafficking. Fluorescent labeling of these APP(BCNK) variants with a membrane-impermeable tetrazine-cy5 conjugate (to confine labeling to only the subpopulation of APP that has trafficked to the cell surface) produced fluorescent rings at the cell membrane, consistent with specific labeling of APP (Figure 3). Since the three best amber variants performed similarly in both western blot and imaging assays, the present disclosure chose the H609TAG variant — with its BCNK incorporation site furthest removed from any proteolytic cleavage site — to further develop into a de novo generatedAβ reporter.

Example 2

To differentiate fluorescent signals that come from theAβ portion of APP vs fully processed Ab, the present disclosure designed a doubly labeled fluorescent reporter in which theAβ portion of APP is labeled with cy5 via genetic code expansion as described above, and the C-terminus of APP is additionally tagged with enhanced green fluorescent protein (EGFP) (Figure la). Co- localized cy5 and EGFP signals correspond to full-length or partially processed APP in whichAβ is still embedded, while standalone cy5 signals should indicate fully processed Ab. The present disclosure confirmed that APP(H609TAG)-EGFP showed BCNK-dependent expression (Figure 4) and that it can be specifically labeled with tetrazine-cy5. To demonstrate that the reporter can be used to visualize de novo generatedAβ in real time, the present disclosure performed tetrazine-cy5 labeling on APP(H609BCNK)-EGFP at 4 °C to minimize endocytosis (the process of which will initiate APP processing), then allowed labeled APP to be endocytosed and processed andAβ produced over time at 37 °C. Right after tetrazine-cy5 labeling at 4 °C, the present disclosure observed cy5 signal exclusively at the cell surface, with strong co- localization between cy5 and EGFP signals (Figure 5, Oh). After incubation at 37 °C for 2 hours, the present disclosure could observe formation of intracellular fluorescent puncta, consistent with APP getting internalized (Figure 5, 2h). The cy5-labeled puncta are highly specific to transfected cells, suggesting that they are true labels on AP(BCNK)-containing species, not non- specific sticking of probes. The cy5 labeling pattern also indicates that the present disclosure specifically captured post-endocytic APP and its derivatives, as APP(H609BCNK)-EGFP present at the rough ER envelope surrounding the cell nuclei is not labeled with cy5 (yellow arrow. Figure 5, 2h). While the majority of cy5 puncta co-localize with EGFP puncta — indicating that these are intact or partially processed APP that still contains theAβ segment — the present disclosure could readily observe EGFP-only puncta (green arrows) which represent the transmembrane C-terminal domain of APP liberated after the secretase cleavage, as well as cy5-only puncta (white arrows) which represent fully processed amyloid β peptide species. Interestingly, these amyloid species cannot be counterstained with plaque-specific Amylo-Glo, suggesting that they do not yet form plaque ultrastructure in this cellular context. The present disclosure noted that the punctateAβ localization pattern revealed by the reporter is distinctly different from simple diffuse staining observed ifAβ is produced exogenously from an expression plasmid (Figure 6). This highlights the need to studyAβ and other bioactive processed peptides under the physiological conditions in which they are generated. Both post- endocytic pool of APP (coincident EGFP and cy5 signals) and fully processedAβ (cy5 signals with no EGFP) showed strong localizations to the trans-Golgi network (TGN, marked by TGN38 protein marker), with no observable distribution to the late endosomes (marked by Rab7a) nor anywhere outside of the endocytic and retrograde compartments (Figure 7). This is consistent with previous observations in which A β40, the predominant species of Aβ¹⁴, is generated in the trans- Golgi network (TGN) from surface populations of APP that have been endocytosed⁷.

Example 3

To simplify theAβ reporter from a cumbersome two-colour reporter whereAβ location has to be inferred from co-localization, to a single fluorescent readout, the present disclosure combined genetic code expansion labelling of theAβ segment of APP with Forster resonance energy transfer (FRET)-based fluorescence quenching. The present disclosure replaced EGFP of APP(H609TAG)-EGFP with the protein labelling tag HaloTag¹⁵, and confirmed its BCNK- dependent expression (Figure 8). HaloTag can be further derivatized with QSY21, a non- fluorescent acceptor with optimal absorbance for cy5 fluorescence¹⁶. In this molecular beacon configuration, cy5 on theAβ segment of APP (labeled via genetic encoding of BCNK) should be quenched by an intramolecular HaloTag-linked QSY21 , and fluoresces only whenAβ is fully processed out of APP. Interestingly, the molecular beacon design places the FRET donor cy5 on the extracellular side of APP and the FRET acceptor QSY21 anchored to the cytosolic side. While the distance spanning the lipid bilayer (~40 Ź⁷) is within the reported 40-60 Å Forster radius of cy5-QSY21 energy transfer¹⁶, it was unclear whether such transmembrane FRET -based quenching would be sufficient to elicit a monofluorescent readout ofAβ production.

The present disclosure further performed double labelling of APP(H609BCNK)-HaloTag with cy5-tetrazine and QSY21 conjugated to a HaloTag chloroalkane ligand (QSY21-C1), and tested dose-dependent quenching of cy5 via increasing concentrations of QSY21-C1. The present disclosure found that labelling with 300 nM QSY21-C1 resulted in quenching of cy5 fluorescence on APP, confirming that transmembrane FRET-based quenching can occur efficiently (Figure

9). Further incubation of doubly labelled cells at 37 °C for 2 hours to allow post-endocytic APP processing resulted in markedly increase in cy5 fluorescence, the punctate pattern of which is distinct from that of APP-HaloTag, which was immunostained via a C-terminal myc tag (Figure

10). While most cy5 puncta do not overlap with APP, suggesting that they are bona fide fully liberated Aβ peptide, puncta with coincident anti-myc staining on APP and cy5 may indicate molecularly separatedAβ and APP in the same endocytic vesicle that cannot be optically separated due to diffraction-limited imaging.

The present disclosure may be embodied in other specific forms without departing from its structures, methods, or other essential characteristics as broadly described herein and claimed hereinafter. The described embodiments are to be considered in all respects only as illustrative, and not restrictive. The scope of the disclosure is, therefore, indicated by the appended claims, rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope. References

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Claims

Claims

1. A method of producing a modified amyloid precursor protein capable of emitting self- reporting signal comprising the steps of providing polynucleotide template having sequence SEQ ID NO. 1 encoding for an amyloid precursor protein (APP); modifying the polynucleotide template through genetic code expansion to replace a natural amino acid encoded at a predetermined position within a region encoding for an amyloid β peptide in the SEQ ID NO. 1 with an unnatural amino acid; expressing the modified polynucleotide template to obtain the APP carrying the amyloid β peptide with the unnatural amino acid; and anchoring a fluorophore to the unnatural amino acid carried on the APP to acquire the modified APP.

2. The method of claim 1 wherein the predetermined position is any one of H609, H610 and Q611.

3. The method of claim 1 further comprising attaching fluorescent protein to a C-terminus of the modified APP.

4. The method of claim 1 further comprising attaching a protein-based labelling tag to a C- terminus of the modified APP and carrying a quencher on the protein-based labelling tag, wherein the quencher is fashioned to absorb or nullify signal emitted from the fluorophore such that the signal emitted by the fluorophore only becomes detachable upon disassociating the amyloid β peptide from the modified APP.

5. The method of claim l, wherein the unnatural amino acid is bicyclononyne-lysine, cyclopropene-lysine, alkyne-lysine, trans-cyclooctene-lysine, any derivatizable amino acid, or any fluorescent amino acid.

6. The method of claim 1 , wherein the fluorophore has fluorescent spectra ranging from 350-700 nm.

7. The method of claim 1, wherein the fluorophore is Cy2, Cy3, Cy5 or Cy7.

8. The method of claim 3, wherein the fluorescent protein is cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), enhanced orange fluorescent protein (OFP), enhanced green fluorescent protein (eGFP), modified green fluorescent protein (emGFP), enhanced yellow fluorescent protein (eYFP) and/or monomeric red fluorescent protein (mRFP) and/or modified derivatives

9. The method of claim 4, wherein the protein-based labelling tag is BCCP (Biotin Carboxyl Carrier Protein), Glutathione-S-transferase-tag, Halo-tag, Maltose binding protein-tag, Nus-tag, Thioredoxin-tag, Fc-tag, SNAP-Tag, or CLIP-Tag.

10. The method of claim 4, wherein the quencher is Dabcyl, Malachite green, TAMRA, BHQ-1, BHQ-2, BHQ-3, Iowa Black, or any of QSY series.

11. A modified amyloid peptide comprising: an extracellular domain; a transmembrane domain; a cytoplasmic domain having a C-terminus being further extended from the transmembrane domain; an amyloid β peptide segment interposing in between the extracellular domain and the transmembrane domain, the amyloid β peptide segment comprising an unnatural amino acid anchored with a fluorophore capable of constantly emitting a detectable signal, wherein the unnatural amino acid is introduced by replacing a natural amino acid found at position H609, H610 or Q611 as setting forth in SEQ ID NO. 2

12. The modified amyloid peptide of claim 11 further comprising a fluorescent protein attached to the C-terminus.

13. The modified amyloid peptide of claim 11 further comprising a protein-based labelling tag attached to the C-terminus and a quencher being anchored to the protein-based labelling tag, the quencher being fashioned to absorb or nullify the signal emitted from the fluorophore such that the signal emitted by the fluorophore only becomes detachable upon disassociating the amyloid β peptide from the modified APP.

14. The modified amyloid peptide of claim 11, wherein the unnatural amino acid is bicyclononyne-lysine, cyclopropene-lysine, alkyne-lysine, trans-cyclooctene-lysine, any derivatizable amino acid, or any fluorescent amino acid.

15. The modified amyloid peptide of claim 11 , wherein the fluorophore has fluorescent spectra ranging from 350-700 nm

16. The modified amyloid peptide of claim 11 , wherein the fluorophore is Cy2, Cy3, Cy5 or Cy7.

17. The modified amyloid peptide of claim 12, wherein the fluorescent protein is cyan fluorescent protein (CFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), enhanced orange fluorescent protein (OFP), enhanced green fluorescent protein (eGFP), modified green fluorescent protein (emGFP), enhanced yellow fluorescent protein (eYFP) and/or monomeric red fluorescent protein (mRFP) and/or modified derivatives

18. The modified amyloid peptide of claim 13, wherein the protein-based labelling tag is BCCP (Biotin Carboxyl Carrier Protein), Glutathione-S-transferase-tag, Halo-tag, Maltose binding protein-tag, Nus-tag, Thioredoxin-tag, Fc-tag, SNAP-Tag, or CLIP-Tag.

19. The modified amyloid peptide of claim 13, wherein the quencher is Dabcyl, Malachite green, TAMRA, BHQ-1, BHQ-2, BHQ-3, Iowa Black, or any of QSY series.