Luciferase-intercalating dye conjugates for ratiometric nucleic acid detection

Abstract

A probe for detecting and quantifying double-stranded DNA is disclosed. The probe comprises a luminescence generating domain and an intercalating fluorescent dye. The probe can be used in a quantitative DNA amplification reaction. Further, a method for detecting or quantifying double-stranded DNA using the probe described herein is disclosed. Also described is the use of the probe in detecting or quantifying DNA.

Description

[Technical field]

[0001] The present invention relates to probes for detecting and / or quantifying double-stranded DNA, in particular to luminescent intercalating probes and methods for detecting or quantifying double-stranded DNA using such probes. [Background technology]

[0002] (Introduction) The detection of DNA and RNA is fundamental for the diagnosis and prevention of infectious diseases caused by pathogens such as viruses and bacteria. The standard for DNA detection is the polymerase chain reaction (PCR), which is based on the rapid generation of large numbers of identical copies from a single fragment of target DNA. PCR relies on the binding of short DNA primers that bind to the target DNA, after which the DNA polymerase enzyme generates new DNA fragments based on the target template. For this purpose, PCR requires three different thermocycling steps that vary the temperature of the reaction mixture between 50-98 °C. To monitor and quantify DNA during PCR, intercalating dye molecules are used that exhibit a large increase in fluorescence when bound to DNA. The fluorescent signal is generated using a light excitation source such as a laser or LED, and the intensity of the collected light is then used as a measure of the DNA concentration. Quantitative PCR (qPCR) has been shown to be able to detect DNA with high sensitivity, but the required setup can be prohibitive in situations where the use of expensive dedicated equipment (including lasers, optics, and temperature controllers) is limited or undesirable, such as point-of-care diagnostic tests. Furthermore, fluorescence suffers from a high background signal, which is problematic when measurements are made in complex media such as a patient's blood.

[0003] To reduce the complexity of PCR-based detection strategies and move them to point-of-care applications, several alternative approaches have been investigated. An established strategy is to use isothermal nucleic acid amplification methods that allow amplification at a constant temperature, thus eliminating the need for thermal cycling equipment. Two recent examples that have attracted attention are the SHERLOCK and DETECTR platforms, which combine isothermal amplification methods with CRISPR-associated (Cas) proteins that can recognize highly specific DNA or RNA sequences. (Gootenberg, JSet al. Nucleic acid detection with CRISPR-Cas13a / C2c2. Science. 356, 438-442 (2017); Chen, JSet al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 360, 436-439 (2018)). The SHERLOCK method uses isothermal amplification by recombinase polymerase amplification (RPA), which utilizes three enzymes for primer binding and subsequent DNA amplification. It can be performed at a constant temperature of 37-42°C, eliminating the need for thermal cycling. Similarly, DETECTR uses loop-mediated isothermal amplification (LAMP), which is performed at a constant temperature of 60-65°C and is based on a looped set of primers specifically designed to facilitate amplification cycles. In both SHERLOCK and DETECTR, a fluorescent output signal is generated when Cas proteins bind to single-stranded RNA and double-stranded DNA, respectively, cleaving a single-stranded nucleic acid reporter that contains a fluorophore and quencher pair. For point-of-care applications, a colorimetric alternative to the fluorescent readout has been realized and incorporated into a lateral flow assay. (Patchsung,M.et al.Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA.Nat.Biomed.Eng.4,1140-1149(2020)).Although these assays successfully combine isothermal amplification with specific and ultrasensitive detection of viral nucleic acid sources, they rely on fluorescent readouts that require external excitation sources or lateral flow strips and generally only provide qualitative information.

[0004] Bioluminescence-based sensors are a powerful alternative strategy to fluorescence detection. By using bioluminescent luciferase enzymes that generate light without the need for external excitation, it is possible to avoid high background signals and the use of specialized optical instruments for detection. Such bioluminescent proteins can be coupled to acceptors to allow bioluminescence resonance energy transfer (BRET), similar to the more commonly used FRET-based approach. This principle has already been employed to create analogues of fluorescence detection methods, such as bioluminescent molecular beacons consisting of a luciferase and an acceptor dye attached to a DNA hairpin. The hairpin initially brings both components into close proximity, facilitating BRET, but separates them upon complexation with the target ssDNA or ssRNA. (Engelen, W., van de Wiel, KM, Meijer, LHH, Saha, B. & Merkx, M. Nucleic acid detection using BRET-beacons based on bioluminescent protein-DNA hybrids. Chem. Commun. 53, 2862-2865 (2017)). Intercalating dyes have also been used in combination with bioluminescent proteins in which luciferase is fused to a DNA-targeting zinc finger protein. (Yoshida et al., Anal. Chem. 2013, 85, 13, 6485-6490). When the zinc finger binds to the target dsDNA, the intercalating dye and luciferase protein come into close proximity, resulting in BRET from luciferase to the dye. Unlike intercalating dyes in qPCR, all of these approaches focus on the specific detection of DNA and require chemical synthesis of a new probe for each new target DNA.

[0005] To improve the sensitivity for point-of-care diagnostic applications, bioluminescence has also been combined with isothermal amplification of target nucleic acids. "Bioluminescence assay in real-time" (BART) relies on LAMP amplification of target DNA and an enzyme that consumes or converts one of the by-products of DNA polymerization to generate light. (Gandelman,OA et al. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time. PLoS One 5, (2010)). This method has been shown to be capable of detecting single copies of DNA, but suffers from autoinhibition, making the output signal time-dependent and difficult to interpret. Furthermore, detection of specific DNA sequences has been explored in combination with isothermal amplification with split luciferase bound to single-stranded DNA (ssDNA). (Chang,D.,Kim,KT,Lindberg,E.& Winssinger,N.Smartphone DNA or RNA Sensing Using Semisynthetic Luciferase-Based Logic Device.ACS Sensors 5,807-813(2020)). Herein, we demonstrate that repeated target sequences on the amplicons of rolling circle amplification (RCA) are recognized by the system, triggering the tandem assembly of split luciferases to reconstitute NanoLuc, resulting in blue light emission. Similar to molecular beacons and zinc fingers, this strategy requires the chemical synthesis of a new probe for every new target DNA.

[0006] These problems are overcome by the present invention as claimed in the accompanying claims. Summary of the Invention

[0007] Bioluminescence-based readouts avoid the problem of requiring complex optical equipment based on lasers or LEDs and employ the luciferase enzyme, which generates light without the need for external excitation. We present a new bioluminescent sensor protein based on a thermostable luciferase chemically conjugated with an intercalating dye, which combines highly sensitive non-specific detection of double-stranded DNA with a simple blue-to-green bioluminescent readout. Thus, our invention allows quantitative and sensitive double-stranded DNA detection without complex and highly specialized equipment, with a convenient optical readout that can be recorded with a simple digital camera or smartphone.

[0008] Thus, in a first aspect, there is provided an intercalating dye probe for the detection of double-stranded DNA, the dye comprising a photoprotein linked to one or more intercalating fluorescent dyes.

[0009] In a second aspect, there is provided a method for detecting double-stranded DNA in a solution, the method comprising incubating an intercalating dye probe according to the first aspect of the invention and a suitable substrate for a luminescent domain with the solution and detecting light emitted by the intercalating fluorescent dye.

[0010] In a third aspect, there is provided a kit of parts comprising an intercalating dye probe according to the first aspect of the invention and a suitable substrate for a luminescent domain.

[0011] In a fourth aspect, there is provided the use of an intercalating dye probe according to the first aspect of the invention in a method for the detection of double-stranded DNA. [Brief description of the drawings]

[0012] [Figure 1]Schematic of bioluminescent intercalating dyes. The probe consists of NanoLuc luciferase (blue, λmax ~ 460 nm) conjugated to an intercalating dye (green) via cysteine-maleimide chemistry. In the absence of double-stranded (dsDNA), there is little fluorescence of the intercalating dye and the blue emission of NanoLuc can be seen (left). In the presence of dsDNA, the intercalating dye binds to dsDNA, BRET occurs, and the dye emits green light (right). [Diagram 2] Figure 2(A) shows the bioluminescence titration of NanoLuc-Thiazole Orange (D148C, flexible loop) with dsDNA. Figure 2(B) shows the bioluminescence titration of NanoLuc-Thiazole Orange (G182C, C-terminus) with dsDNA. For both mutants: the complex (left, 1 nM) was added to a 2-fold dilution series of salmon sperm DNA from 39 μM to 10,000 μM (in DNA base pairs). Incubation was performed at room temperature for 30 min in 1xPBS + 1 mg / mL BSA, 5% DMSO, pH 7.4. After addition of NanoGlo substrate (1000-fold dilution), the luminescence intensity was measured at 398-653 nm. [Figure 3-1] Figure 3(A) is a schematic diagram of various linker mutants containing one to three lysines and two glycines (negative control) incorporated into the C-terminus of NanoLuc. [Figure 3-2]Figure 3(B) shows the bioluminescence titration with dsDNA of NanoLuc variants with two intercalating dyes and one (NL2C-1K), two (NL2C-2K) or three (NL2C-3K) lysines in between (at G182, C-terminus). Figure 3(C) shows the bioluminescence titration with dsDNA of NanoLuc variants with two intercalating dyes and two lysines (NL2C-2K) or two glycines (NL2C-2G, negative control) in between (at G182, C-terminus). For all variants: complexes (1 nM) were added to a two-fold dilution series of salmon sperm DNA from 0.76 μM to 5,000 μM (in DNA base pairs). Incubation was performed for 30 min at room temperature in 1xPBS + 1 mg / mL BSA, 5% DMSO, pH 7.4. After addition of NanoGlo substrate (1000-fold dilution), the emission intensity was measured at 398-653 nm. [Figure 4] Figure 4(A) is a schematic of a NanoLuc variant containing two cysteines separated by two positively charged lysine residues. Figure 4(B) is a schematic of a two-dye Nanoluc-2xTO probe. This probe consists of NanoLuc luciferase (blue) bound to two intercalating dyes (green) separated by a positively charged linker, which is expected to interact with the negatively charged dsDNA backbone. [Diagram 5]Figure 5 (B,D) shows the bioluminescence titration of NanoLuc-2xTO with dsDNA with 2xTO at the C-terminus (B) or flexible loop (D). The complex (1 nM) was added to a 3-fold dilution series of salmon sperm dsDNA ranging from 0.76 μM to 5000 μM base pairs. Incubation was performed at room temperature for 30 min in 1xPBS + 1 mg / mL BSA, 5% DMSO, pH 7.4. After addition of NanoGlo substrate (1000-fold dilution), the emission intensity was measured at 398-653 nm. Technical duplicates are represented by circles, and dashed lines connect the average values. Figure 5 (A,C) shows the full emission spectrum of the bioluminescence titration of NanoLuc-2xTO with dsDNA with 2xTO at the C-terminus (A) or flexible loop (C). Data points represent average values. *Data in Panel B are the same as those in Figure 3BC (2-lysine linker, blue curve). [Figure 6] Comparison of bioluminescence titration of NanoLuc-1xTO (green), NanoLuc-2xTO (blue), and NanoLuc-3xTO (red) with dsDNA. Complexes (1 nM) were added to a 3-fold dilution series of salmon sperm dsDNA ranging from 0.76 μM-5000 μM base pairs. Incubation was performed for 30 min at room temperature in 1xPBS + 1 mg / mL BSA, 5% DMSO, pH 7.4. After addition of NanoGlo substrate (1000-fold dilution), luminescence intensity was measured from 398 to 653 nm. Technical duplicates are represented by circles and dashed lines connect the mean values. [Figure 7-1] Figure 7(A) is a schematic of the conjugation process involving a single cysteine ​​NanoLuc variant, a 1-(2-aminoethyl)maleimide crosslinker, and an NHS-activated intercalating dye, while Figure 7(B) is a reaction scheme for coupling of the maleimide-activated crosslinker with NHS-TO. [Figure 7-2] Figure 7(C) shows the LCMS results of NHS-TO before (top) and after (bottom) crosslinker coupling. The molecular weights are indicated on top of the chromatogram peaks. [Figure 8]Q-ToFLC-MS spectra of NanoLuc variants with one cysteine ​​introduced at the C-terminus before (gray, NL2) and after (black, NL2+TO) dye coupling. The peaks at +18 and +36 can be attributed to hydrolysis of the maleimide moiety and photodetachment of the dye. Average molecular weight NL2=22145.3. Calculated average molecular weight NL2+TO=22672.2. [Figure 9] Q-ToFLC-MS spectra of NanoLuc variants incorporating two cysteines at the C-terminus before (red, NL2xCYS) and after (black, NL2xCYS+TO) dye coupling. The peaks at +18, +36 may be due to hydrolysis of the maleimide moiety and photodegradation of the dye. The peak at -102 may be due to oxidative cleavage or by-products from the binding of lysine residues with the NHS-intercalated dye. Calculated average molecular weight NL2xCYS=22367.6. Calculated average molecular weight NL2+TO=23422.4. [Figure 10]Development of a two-stage assay for SARS-CoV-2 cDNA detection. Figure 10A is a schematic of the two-stage assay setup. SARS-CoV-2 cDNA is amplified by LAMP for 35 min at 65 °C and incubated at room temperature for 30 min in combination with 1 nM LUMID-2F sensor, followed by addition of furimazine (1000-fold dilution) and measuring bioluminescence using a plate reader and / or a smartphone camera. Figure 10B shows the sensor response of the two-stage assay using a plate reader for detection. Data represent technical replicates, n = 3 independent cDNA preparations. Top: Green / blue ratios at different cDNA input concentrations ranging from 2 aM (1.2 copies / μL) to 200 fM (120,000 copies / μL). Green / blue ratios were calculated by dividing bioluminescence at 533 nm by emission at 458 nm and expressed as mean ± standard deviation. Bottom: Total emission spectra represented for buffer (gray), LAMP reaction without target cDNA (NTC, blue), and LAMP reaction with 200 aM target cDNA (green). RLU (relative luminescence units) were normalized to the NanoLuc peak at 458 nm, and the spectra (bottom) are expressed as mean ± standard deviation. Figure 10C shows the sensor response of the two-step assay using a smartphone (Xiaomi mi 9 lite) in a dark styrofoam box. The pictures show the sensor output for different input concentrations of cDNA ranging from 2 aM (1.2 copies / μL) to 200 fM (120,000 copies / μL). Each column represents a technical replicate, with n = 3 independent cDNA preparations. The same samples were detected by the smartphone camera (right) and the plate reader (left). Figure 10D shows the correlation of the green / blue ratio calculated from the plate reader and the smartphone camera. [Figure 11]Development of a one-pot assay for cDNA detection of SARS-CoV-2. Figure 11A is a schematic of the one-pot assay setup. All reaction components (SARS-CoV-2 cDNA, LUMID-2F sensor, NanoLuc substrate, LAMP reaction components) are mixed in one tube and incubated at 65 °C for 35 min. After cooling, the results are read directly using a plate reader and / or a smartphone camera. Figure 11B shows the sensor response of the one-pot assay, using a plate reader for detection. Data represent technical replicates, n = 3 independent cDNA preparations. Left: Green / blue ratios at varying cDNA input concentrations ranging from 20 aM (12 copies / μL) to 2 fM (1,200 copies / μL). Green / blue ratios were calculated by dividing bioluminescence at 533 nm by emission at 458 nm and expressed as mean ± standard deviation. Circles indicate individual data points. Right: Total emission spectra represented for buffer (gray), LAMP reactions without target cDNA (NTC, blue), and LAMP reactions with 200 aM target cDNA (green). Spectra (bottom) are presented as mean ± standard deviation. Figure 11C shows the sensor response of a one-step assay using a conventional digital camera (Sony DSC-RX100) in a dark Styrofoam box. The pictures show the sensor output for different input concentrations of cDNA ranging from 20 aM (12 copies / µL) to 2 fM (1,200 copies / µL). Each column represents a technical replicate, with n = 3 independent cDNA preparations. The same samples were detected with a smartphone camera (right) and a plate reader (left). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention seeks to develop a new class of bioluminescent sensor proteins that combine sensitive nonspecific detection of DNA with a simple camera-based readout suitable for point-of-care diagnostics. To this end, we have chemically coupled one or more intercalating dyes to the thermostable NanoLuc luciferase, which emits blue light bioluminescence upon addition of substrate. In the absence of dsDNA, the intercalating dye emits minimal fluorescence and only the blue light of NanoLuc is seen. In the presence of dsDNA, the intercalating dye binds to dsDNA, allowing bioluminescence resonance energy transfer (BRET) from NanoLuc to the dye, resulting in the emission of green light (Figure 1). The ratio of the emission of NanoLuc to that of the intercalating dye allows the amount of dsDNA in a sample to be measured. We aim to combine these probes with isothermal amplification strategies such as RPA and LAMP to provide a preamplification step to further increase the sensitivity of the assay. This approach allows for simple and sensitive DNA detection without the use of high-tech equipment and external excitation sources, opening up the possibility of point-of-care applications where access to resources or trained staff may be limited.

[0014] Thus, a simple method for the detection of DNA is provided that does not require specialized personnel or equipment, and can be applied in the diagnosis and prevention of infectious diseases. The present invention describes a novel molecular probe consisting of two parts: a light-emitting bioluminescent protein directly bound to an intercalating dye. When the probe binds to DNA, the two interact and the color of the light produced changes, for example, from blue to green. After simply adding the probe to a sample, the color change indicating the presence of DNA can be recorded and quantified using a simple camera or smartphone.

[0015] Thus, in a first aspect, an intercalating dye probe for detecting double-stranded DNA is provided, which comprises a luminescent protein bound to one or more intercalating fluorescent dyes. The luminescent protein may be a bioluminescent protein or an enzyme used to enhance chemiluminescence.

[0016] As used herein, luciferase refers to a protein capable of generating bioluminescence in the presence of a suitable substrate. Such proteins can be naturally occurring proteins (e.g., proteins isolated from organisms that exhibit bioluminescence) or modified or synthetic variants thereof. It is understood that luciferase refers to a wide variety of unrelated proteins that have in common the ability to exhibit bioluminescence (emitting light in the presence of a substrate), and thus any protein that can exhibit bioluminescence is considered a bioluminescent protein for the purposes of the present invention. As used herein, luciferase activity refers to the ability of a protein to convert a substrate resulting in light emission (bioluminescence). Thus, in one embodiment, the bioluminescent protein is a protein with luciferase activity, preferably selected from nanoluc, firefly luciferase, Renilla luciferase, copepod luciferase, bacterial luciferase, dinoflagellate luciferase, deep-sea shrimp luciferase, Gaussia luciferase, TurboLuc luciferase, Aluc luciferase, or catalytically active fragments thereof.

[0017] The bioluminescent protein is directly linked to the intercalating dye probe, allowing the transfer of emitted light from the bioluminescent protein to the intercalating dye, resulting in excitation and subsequent fluorescence of the dye. Because the dye only exhibits fluorescence when intercalated into double-stranded DNA, the dye-specific emission can be used to detect or quantitate double-stranded DNA (dsDNA), as described herein below.

[0018] As used herein, "linked" refers to a covalent bond. Methods for linking dyes to proteins are widely known in the art. For example, dyes may be linked to cysteine ​​residues of proteins, as described below. For example, if a suitable cysteine ​​is not available, a cysteine ​​may be introduced into the protein by point mutation. It is further understood that multiple cysteines may be introduced to allow linking to multiple dyes. Preferably, the cysteines are spatially separated by being introduced into different regions of the protein or by including a linker sequence, for example a one-lysine linker, a two-lysine linker or a three-lysine linker. Additionally, cysteines present at inappropriate positions in the protein may be removed by point mutation. Thus, in one embodiment, a protein having luciferase activity comprises an amino acid sequence defined by any one of SEQ ID NOs: 1-7.

[0019] Summary of sequences used in this application: [Table 1] The protein sequence is start codon (Met, first amino acid); Streptavidin (STREP) tag-(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys; amino acids 2–9); The modified NanoLuc protein sequence (starting at amino acid 10); and Contains a 6-histidine tag (the last 6 amino acids).

[0020] The numbering of the mutations in the above sequence follows the native nanoluc sequence and therefore starts from the methionine at position 10 of the presented sequence, excluding the STREP tag.

[0021] Alternatively, the dye may be conjugated to an enzyme that generates a substrate that allows for chemiluminescence. For example, horseradish peroxidase may be conjugated to allow for catalysis of the oxidation of luminol with hydrogen peroxide. Thus, in one embodiment, the enzyme used to enhance chemiluminescence is selected from horseradish peroxidase and alkaline phosphatase.

[0022] Suitable intercalating fluorescent dyes are known to those skilled in the art. In one embodiment, the intercalating fluorescent dye is selected from acrisin orange, thiazole orange, ethidium bromide, SYBR Green I, SYBR Gold, SYBR Safe, EvaGreen, EvaRuby, PicoGreen, SYTO-9, TOTO-1, and YOYO-1. It is understood that the intercalating dye probe may include one or more intercalating fluorescent dyes, such as one, two, three, four, five or more. Multiple fluorescent intercalating dyes may be added to enhance the DNA targeting of the probe. In one embodiment, the intercalating dye probe further includes an additional DNA binding domain. This may be advantageous to further enhance DNA binding or targeting. In a second aspect, the present invention relates to a method for detecting double-stranded DNA in a solution, the method comprising the steps of: Incubating a solution of an intercalating dye probe according to the first embodiment and a suitable substrate for a luminescent domain; and detecting light emitted by the intercalating fluorescent dye.

[0023] Since the intercalating dye is excited by the luminescent protein only when it is intercalated into double-stranded DNA, dsDNA can be detected by detecting the light emitted by the dye. Also, the amount of dsDNA can be quantified by detecting the light emitted by the luminescent protein (e.g., directly by a bioluminescent protein or indirectly by an enzyme that enhances chemiluminescence). Thus, in one embodiment, the method is used to quantify double-stranded DNA, and the method includes: detecting light emitted by the light-emitting domains; and quantifying the amount of double-stranded DNA based on the ratio of the light emitted by the intercalating fluorescent dye and the light emitted by the light-emitting domain. The method can be performed before, during, and / or after the DNA amplification reaction. The method can be performed once or continuously during the amplification reaction.

[0024] It is understood that the method may be performed with any type of amplification reaction that amplifies dsDNA. In one embodiment, the amplification reaction is selected from polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification (HDA) or divergent amplification (RAM).

[0025] In a third aspect, the present invention relates to a kit of parts comprising the intercalating dye probe according to the first aspect of the present invention and a suitable substrate of luminescent domain.In one embodiment, the substrate is luciferin, preferably furimazine, firefly luciferin, Latia (snail) luciferin, bacterial luciferin, coelenterazine, dinoflagellate luciferin, vargulin, 3-hydroxyhispidin or luminol.It is understood that luciferin is a general term for compounds that act as substrates for luciferase, and thus luciferin herein refers to any suitable substrate of luciferase as defined herein.

[0026] In a fourth aspect, the present invention relates to the use of an intercalating dye probe according to the first aspect of the invention in a method for the detection of double-stranded DNA.

[0027] Other variations to the disclosed implementations can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. EXAMPLES

[0028] (material and method) Cloning The pET28a vector containing DNA encoding NanoLuc luciferase with a Strep-tag at the N-terminus and a hexahistidine-tag at the C-terminus was ordered from GenScript. Site-directed mutagenesis was performed to mutate the native cysteine ​​to a serine (C166S) and introduce new cysteine ​​and lysine residues using specific primers according to the manufacturer's instructions with the QuikChange Lightning Site-Directed Mutagenesis kit (Agilent). All cloning and mutagenesis results were confirmed by Sanger sequencing (BaseClear).

[0029] Protein expression and purification The plasmid encoding NanoLuc was transformed into chemically competent E. coli BL21(DE3) and cultured in 2YT medium (16 g peptone, 5 g NaCl, 10 g enzyme extract per liter) supplemented with 50 μg / ml kanamycin. OD 600 Protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at pH 7.0 = 0.6 overnight at 20°C. Cells were then harvested by centrifugation and lysed using Bugbuster protein extraction reagent (Novagen) supplemented with benzonase endonuclease (Novagen). 2+The protein was purified by -NTA affinity chromatography, and the eluted fraction was exchanged into storage buffer (100 mM Tris-HCl, 150 mM NaCl, pH 8.0). The purity and accurate mass of the protein were confirmed by SDS-PAGE and Q-ToF LC-MS, respectively. The purified protein was stored at -80°C until conjugation.

[0030] Conjugation of NHS-activated dyes with 1-(2-aminoethyl)maleimide crosslinker Amine-reactive N-hydroxysuccinimide (NHS) esters of thiazole orange (TO) and acrisin orange (AO) were obtained from Biotium and dissolved in DMSO to a final concentration of 20 mM each. Then, 3 equivalents of N,N-diisopropylethylamine (DIPEA) and 0.8 equivalents of 1-(2-aminoethyl)maleimide crosslinker (Sigma Aldrich) were added and incubated overnight at room temperature with continuous shaking at 450 rpm (Figure 7B). The correct mass after conjugation was confirmed using LC-MS (Figure 7C). The maleimide-activated dyes were stored at -30 °C until further use.

[0031] Conjugation of maleimide-activated dyes with NanoLuc Prior to conjugation of NanoLuc to the maleimide-activated dye, the protein was first reduced by incubation with 5 mM TCEP for 1 h at room temperature with continuous shaking at 500 rpm, followed by buffer exchange into sodium phosphate buffer (100 mM NaPi, 25 μM TCEP, pH 7.0) using a PD-10 desalting column (GE Healthcare). Next, both maleimide-activated TO and AO were added in 10-fold molar excess to 10 μM reduced NanoLuc and reacted for 2 h at room temperature with continuous shaking at 500 rpm. The NanoLuc-dye conjugate was purified with a PD-10 desalting column to remove excess dye and was simultaneously buffer exchanged into PBS (100 mM NaPi, 150 mM NaCl, pH 7.2). The dye-protein conjugate was excited with blue light at 470 nm, and the coupling and purification were checked using SDS-PAGE. Finally, the coupling efficiency and correct mass of the NanoLuc-dye conjugates were confirmed by Q-ToF LC-MS.

[0032] Bioluminescence assay Bioluminescence assays were performed in PerkinElmer flat white 384-well Optiplates at a sensor protein concentration of 1 nM in a total volume of 20 μL. Fragmented salmon sperm dsDNA fragments of >2000 bp were ordered from Thermo Fisher and diluted to a concentration range of 0.7 μM to 5 mM (measured in number of base pairs). The sensor protein and dsDNA fragments were incubated at room temperature for 0.5 h before adding NanoGlo substrate (Promega, N1110) at a final dilution of 1:1000. Bioluminescence spectra were recorded on a plate reader (Tecan Spark 10M) with a step size of 15 nm, a bandwidth of 25 nm, and an integration time of 250 ms to record the entire spectrum between 398 nm and 653 nm.

[0033] Two-step SARS-CoV-2 cDNA assay LAMP primers targeting the cDNA sequence of the SARS-CoV-2 virus nucleocapsid (N) gene were designed using the NEB LAMP Primer Design Tool and ordered from IDT. The sequence of the N gene can be found in SEQ ID NO:8. Primers were diluted to a 10x concentrated stock containing 16 μM inner primer, 2 μM outer primer, and 4 μM loop primer. A plasmid containing the SARS-CoV-2 cDNA sequence was obtained through the Free Genes Project, and the nucleocapsid (N) gene sequence was PCR amplified from this plasmid using specific primers. The N gene target DNA was serially diluted to a 25x concentrated stock ranging from 5 aM to 5 pM. A positive LAMP reaction was performed in a UV PCR cabinet with 1x isothermal amplification buffer (NEB), 6 mM MgSO4 (NEB), 1.4 mM dNTPs (NEB), 1x LAMP primer mix, and 1x target DNA in a total volume of 24 μL. For the no-template controls, similar conditions were used, only the target DNA was replaced with MilliQ water. Reactions were kept on ice during the entire assembly process. To initiate the reaction, Bst 2.0 polymerase (8U, NEB) was added, followed by incubation at 65 °C for 35 min. Then, 10 μL of each LAMP reaction was combined with 10 μL of LUMID-2F sensor (2 nM in PBS buffer (pH 7.4, 0.1% (w / v) BSA, 5% DMSO)) in a PerkinElmer flat white 384-well Optiplate. As a negative control, the LAMP reaction was substituted with PBS buffer. After 30 min of incubation at room temperature, NanoGlo substrate (Promega, N1110) was added at a final dilution of 1:1000. Emission spectra were recorded from 398 nm to 653 nm on a plate reader (Tecan Spark 10M) with a step size of 15 nm, a bandwidth of 25 nm, and an integration time of 100 ms. The green / blue ratio was calculated by dividing the bioluminescence at 533 nm by the emission at 458 nm. The luminescence signal was also recorded with a smartphone (Xiaomi mi 9 lite) camera through a hole in a styrofoam box to exclude ambient light.The photo was taken with an exposure time of 32 seconds and an ISO value of 3200.

[0034] One-pot SARS-CoV-2 cDNA assay The LAMP reaction was constructed as described for the two-step assay, but with the addition of 1 μL of LUMID-2F sensor (25 nM in PBS buffer (pH 7.4, 0.1% (w / v) BSA, 5% DMSO)) and 1 μL of NanoGlo substrate (1:40 dilution). To initiate the reaction, Bst2.0 polymerase (8 U, NEB) was added, followed by incubation at 65 °C for 35 min. Real-time luminescence was monitored using a digital camera (SONY DSC-RX100) through a hole in a polystyrene foam dark box that contained a heating plate for maintaining the reaction temperature. After incubation, the reaction was transferred to room temperature and cooled for 5 min before recording the luminescence signal. Emission spectra were recorded on a plate reader (Tecan Spark 10M) from 398 nm to 653 nm with a step size of 15 nm, a bandwidth of 25 nm, and an integration time of 100 ms. The green / blue ratio was calculated by dividing the bioluminescence at 533 nm by the emission at 458 nm. The luminescence signal was recorded using a setup for real-time luminescence monitoring. The pictures were taken with an exposure time of 30 seconds and an ISO value of 6400.

[0035] (Results and Discussion) Single-dye NanoLuc variants The synthesis of luciferase-intercalating dye probes was performed by conjugating single-cysteine ​​NanoLuc variants to the NHS-activated forms of the intercalating dyes thiazole orange (TO) or acrisin orange (AO) via a heterobifunctional 1-(2-aminoethyl)maleimide crosslinker. TO was chosen due to its large increase in fluorescence upon complexation with dsDNA (>3000-fold), its suitable spectral overlap with NanoLuc (em. Nanoluc 460 nm, ex. TO 514 nm), and the commercial availability of its NHS-activated form. AO is an alternative intercalating dye that has been used as a probe to discriminate between double-stranded and single-stranded nucleic acids. AO is already fluorescent in solution, and although the intrinsic increase in fluorescence upon dsDNA binding is small, its strong intrinsic fluorescence was utilized to analyze the conjugation procedure. To allow site-specific conjugation, cysteine ​​mutations were introduced in the flexible loop region (D148) and C-terminus (G182) of NanoLuc, known to be suitable positions for conjugation without inhibiting the bioluminescent properties of NanoLuc. A hexahistidine tag at the C-terminus was included to facilitate purification of single-cysteine ​​NanoLuc mutants (the DNA sequence, amino acid sequence and protein mutants are listed in the table above). First, a 1-(2-aminoethyl)maleimide crosslinker was conjugated to an NHS-activated intercalating dye, and the reaction product was then conjugated to NanoLuc (Figure 3, Figure 7). Q-ToF LC-MS analysis revealed that the synthesis was successful, although minor peaks due to photobleaching and hydrolysis of the maleimide functional group were observed (Figure 8).

[0036] To investigate the analytical performance of the NanoLuc-thiazole orange probe for dsDNA, a bioluminescence titration with dsDNA was performed (Figure 2). The results revealed that both probes can intercalate into dsDNA, as the emission of green light (~533 nm) increased with increasing amounts of dsDNA (Figure 2A, center). Moreover, the ratio of the emission of thiazole orange (533 nm) to that of NanoLuc (458 nm), or the "BRET ratio", increased with increasing concentrations of dsDNA, further supporting the dsDNA dependence of the sensor (Figure 2A, right). Nevertheless, BRET only appeared at high concentrations of dsDNA (~mM range), indicating a low affinity of the probe for dsDNA. The increase in the BRET ratio with dsDNA addition was larger for constructs with the dye located at the flexible loop region (Figure 2A, 4-fold) compared to constructs with the dye located at the C-terminus (Figure 2B, <2-fold). Since BRET is strongly distance-dependent, this difference is likely due to the distance between the dye and the active site of NanoLuc in both mutants, i.e., placing the dye at the flexible C-terminus (G182C) increases the distance to the active site compared to the D148C mutant, and therefore reduces the BRET efficiency.

[0037] Multi-dye NanoLuc variants - linker optimization A promising engineering strategy to increase the overall affinity of the probe is to introduce multivalency into the current system by incorporating multiple dyes. The development of a dimeric thiazole orange dye (TOTO) has shown an increase in affinity for dsDNA of about three orders of magnitude compared to its monomeric counterpart. 13 Similarly, a small peptide containing two Acrysin Orange dyes separated by two lysine residues that add electrostatic interactions with dsDNA was shown to increase the affinity of the dyes by 2–3 orders of magnitude. 14To apply the concept of multiple dyes with positively charged linkers to our probes, we incorporated a second intercalating dye, separated by a lysine residue, in close proximity to the first intercalating dye at the C-terminus of Nanoluc(C180). The amount of lysines was varied from one to three to find the optimal distance at which the dye binds and intercalates into dsDNA, and a glycine residue was incorporated as a control to confirm the necessity of a positive charge (Figure 3A).

[0038] Bioluminescence titrations with dsDNA revealed that the two-lysine linker reacted with the highest affinity and the highest dynamic range to dsDNA (Figure 3B, blue). The variant with the three-lysine linker had a similar affinity but a smaller dynamic range. This is likely because both dyes were unable to effectively intercalate into dsDNA, resulting in less green fluorescence (Figure 3B, red). The lowest affinity and dynamic range was observed with the one-lysine linker. This can be explained by a lesser amount of positive charge as well as a less optimal distance at which the dye binds to dsDNA (Figure 3B, green). Control experiments with the two-glycine linker showed an affinity approximately 100-fold lower than that of the two-lysine linker, indicating that the positive charge is essential for improved dsDNA binding (Figure 3C).

[0039] Multi-dye NanoLuc variants - 2 dyes Next, we engineered two cysteines separated by the best performing two-lysine linker at the C-terminus and flexible loop region of NanoLuc (NanoLuc-2xTO, Figure 4). Q-ToF LC-MS analysis revealed that the coupling reaction was complete, although peaks due to photobleaching and hydrolysis of the maleimide functional group were observed (Figure 9).

[0040] To investigate the analytical performance of the NanoLuc-2xTO sensor towards dsDNA, we again performed a bioluminescence titration with dsDNA (Figure 5). With increasing concentrations of dsDNA, an increase in the ratio between the emission of thiazole orange (533 nm) and that of NanoLuc (458 nm) (BRET ratio) was observed. BRET appeared already in the low μM range, indicating a 1000-fold increase in affinity compared to the sensor variant containing only one TO (compare Figure 2). A moderate increase of 4-fold in the BRET ratio was observed for the variant with 2xTO at the C-terminus and a 9-fold increase for the variant with 2xTO in the flexible loop (compare Figure 5B and D). Similar to the results obtained with the single dye variants in Figure 2, these results indicate that the BRET efficiency is strongly distance dependent and that the BRET efficiency is increased by placing the intercalating dye in the flexible loop.

[0041] Interestingly, the total emission spectrum revealed a positive correlation between the total emission intensity and the amount of dsDNA. It is hypothesized that the addition of the second dye increases the hydrophobicity of the system, thereby decreasing the solubility of NanoLuc-2xTO and decreasing the signal intensity. The large amount of negatively charged dsDNA may have acted to stabilize the protein-dye construct or prevent the formation of aggregates, leading to the observed increase in absolute signal.

[0042] Multi-dye NanoLuc variant-3 dye We also evaluated the performance of the sensor when a third dye, separated by a two-lysine linker, was added to the C-terminus of NanoLuc (NanoLuc-3xTO, Figure 6). From the titration with dsDNA, a lower limit of detection (LoD) and higher affinity were observed compared to the variant with two dyes. Furthermore, the curves are stretched over a wider range of dsDNA concentrations and the fold change in the BRET ratio appears to be smaller. This may indicate the presence of a mixture of different affinity binders, resulting from an incomplete dye coupling reaction. The result was NanoLuc variants with one, two or three dyes that bind to dsDNA in different concentration regions.

[0043] The present invention describes a comprehensive bioluminescent sensor for dsDNA detection, based on one or more intercalating dyes conjugated to a light-emitting luciferase protein. Multivalent DNA binding of one or more dyes per probe, combined with the use of short positively charged lysine linkers between the dyes, resulted in high-affinity DNA detection probes with a simple bioluminescent readout. As a result, dsDNA could be detected with μM-level affinity (measured in base pairs) and BRET ratios changed 4-9 fold. Many amplification methods generate dsDNA fragments of 100-200 bp, suggesting that the sensor developed here can detect such fragments already in the low nM range.

[0044] The present invention envisages its use for detecting viral DNA or RNA in patient samples such as blood or saliva, using rhinoviruses that cause the common cold, as well as the highly related and destructive SARS-CoV-2 virus. To this end, the bioluminescent intercalating proteins described herein can be combined with amplification techniques (e.g., RPA or LAMP), providing a simple way to perform an initial preamplification step to increase the sensitivity of the assay required to directly detect DNA in patient samples. Due to the thermostability of NanoLuc, it is expected that the NanoLuc-TO complex can be used to monitor in real time the progress of RPA (T = 42 °C) in a one-pot reaction. Furthermore, the present invention envisages further increasing the affinity of the luciferase-TO complex for dsDNA by combining a bivalent TO label at the loop position with a bivalent label at the C-terminus. Furthermore, it is envisaged that the sensitivity can be further improved by fusion of the NanoLuc domain to a general dsDNA binding domain.

[0045] Two-step assay for SARS-CoV-2 complementary DNA The dsDNA fragments are typically 100–200 base pairs long, suggesting that the developed sensor can detect such fragments in the low nanomolar range. As many diagnostic applications, such as viral nucleic acid detection, require attomolar sensitivity, we sought to combine the developed bioluminescent probe with various isothermal amplification steps to develop a simple and sensitive assay platform that can be used at the point-of-care. As a result, we found that the LAMP method was most suitable due to its high yield of dsDNA while minimizing nonspecific amplification (avoiding large background signals is essential when employing nonspecific readout). Inspired by the ongoing COVID-19 pandemic, we designed a LAMP reaction to target the complementary DNA (cDNA) sequence of the nucleocapsid (N) gene of the SARS-CoV-2 virus and explored the feasibility of LUMID probes in rapid point-of-care detection for virus detection.

[0046] First, we developed a two-step assay in which serially diluted SARS-CoV-2 cDNA was amplified by the LAMP method and then combined with LUMID-2F to facilitate bioluminescence detection (Figure 10A). For this purpose, the LAMP reaction was performed for 35 min at 65 °C according to the manufacturer's instructions and then combined with 2 nM of sensor protein at 1:1 (v / v). After 30 min of incubation and addition of substrate, DNA concentrations up to 200 aM (120 copies / µL) could be distinguished from the non-template control (NTC) by a 2.5-fold change in the green / blue emission ratio (Figure 10B). To demonstrate the potential for point-of-care applications, the signal was recorded in a styrofoam dark box using a standard smartphone camera (Xiaomi mi 9 lite). Photographs of the same samples used for the plate reader measurements visually confirmed a clear color change from blue to green for all DNA concentrations up to 200 aM (Figure 10C). The exact emission ratio was calculated from the blue and green channels of the smartphone RGB image and showed a linear correlation with the ratio obtained from the plate reader measurement (Figure 10D), with a Pearson coefficient of 0.996. This revealed that the performance of the smartphone camera was comparable to that of the plate reader, although the absolute changes in emission ratios were different. In both detection methods, the amount of green light from the NTC was higher than that of the buffer-only control. This is likely because the long (~40 bp) primers used in the LAMP reaction form secondary dsDNA structures that can be detected without amplification. Although this primer-related background signal reduces the dynamic range of the assay, the observed emission ratio changes are robust and comparable to those of common BRET-based sensors. These results indicate that the LUMID sensor can be combined with LAMP to construct a highly sensitive nucleic acid assay platform that can provide results in about 1 hour by simple detection using a smartphone.

[0047] One-pot assay for SARS-CoV-2 complementary DNA We next assessed whether all assay components could be combined in one tube, demonstrating the viability of this platform as a one-step point-of-care diagnostic tool. This approach not only simplifies the experimental procedure but also reduces the risk of false positives that may arise as a result of cross-contamination during post-amplification reaction transfer. Thus, the LAMP reaction components were combined in one tube with serially diluted SARS-CoV-2 cDNA, LUMID-2F sensor (1 nM), and NanoLuc substrate (1000-fold diluted) and then incubated at 65 °C for 35 min (Figure 11A). NanoLuc was inactive at the reaction temperature of 65 °C, but cooling at room temperature for 5 min fully restored the enzyme activity. With this short cool-down step, DNA concentrations up to 20 aM (12 copies / µL) were discriminated from the non-template control (NTC) with a 2.1-fold change in the green / blue emission ratio (Figure 11B). Additionally, photographs of the same samples used for plate reader measurements showed a visual color change up to a DNA concentration of 20 aM (Figure 11C). This indicates that the transition to a one-pot assay did not compromise sensitivity, maintaining the attomole sensitivity reported in the two-step assay. An apparent 10-fold increase in affinity was observed, but this is likely due to the inherent inter-reaction variability of the LAMP reaction.

Claims

1. 1. An intercalating dye probe for the detection of double-stranded DNA, said dye comprising a luminescent protein bound to one or more intercalating fluorescent dyes.

2. 2. The intercalating dye probe of claim 1, wherein the luminescent protein is a bioluminescent protein or an enzyme used to enhance chemiluminescence.

3. 3. The intercalating dye probe of claim 2, wherein the bioluminescent protein is a protein having luciferase activity, preferably selected from nanoluc, firefly luciferase, Renilla luciferase, copepod luciferase, bacterial luciferase, dinoflagellate luciferase, deep-sea shrimp luciferase, Gaussia luciferase, TurboLuc luciferase, Aluc luciferase, or catalytically active fragments thereof.

4. 4. The intercalating dye probe according to claim 3, wherein the protein having luciferase activity comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 7.

5. 3. The intercalating dye probe of claim 2, wherein the enzyme used to enhance the chemiluminescence is selected from horseradish peroxidase and alkaline phosphatase.

6. 2. The intercalating dye probe of claim 1, wherein the intercalating fluorescent dye is selected from acrisin orange, thiazole orange, ethidium bromide, SYBR Green I, SYBR Gold, SYBR Safe, EvaGreen, EvaRuby, PicoGreen, SYTO-9, TOTO-1, and YOYO-1.

7. The intercalating dye probe of claim 1 further comprising an additional DNA binding domain.

8. 1. A method for detecting double-stranded DNA in a solution, comprising: Incubating said solution with a suitable substrate for the intercalating dye probe and luminescent domain according to any one of claims 1 to 7; detecting light emitted by the intercalating fluorescent dye; A method for detecting double-stranded DNA comprising:

9. It is used to quantify double-stranded DNA, and further detecting light emitted by the light-emitting domain; 9. The method for detecting double-stranded DNA according to claim 8, further comprising a step of quantifying the amount of double-stranded DNA based on the ratio of the light emitted by the intercalating fluorescent dye to the light emitted by the light-emitting domain.

10. 9. The method for detecting double-stranded DNA according to claim 8, which is carried out before, during, and / or after a DNA amplification reaction.

11. 11. The method for detecting double-stranded DNA according to claim 10, wherein the DNA amplification reaction is selected from polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), helicase-dependent amplification (HDA), or divergent amplification method (RAM).

12. A kit of parts comprising an intercalating dye probe according to any one of claims 1 to 7 and a suitable substrate for a luminescent domain.

13. The substrate is a luciferin, preferably furimazine, firefly luciferin, snail luciferin, bacterial luciferin, coelenterazine, dinoflagellate luciferin, vargulin, 3-hydroxyhispidin, luminol or H ２ O ２ 13. The kit of parts according to claim 12, wherein

14. Use of the intercalating dye probe according to any one of claims 1 to 7 in a method for detecting double-stranded DNA.

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