High-fidelity time sequence fluorescent DNA bar code and application thereof

Abstract

The invention provides a high-fidelity time sequence fluorescent DNA bar code and application thereof. The high-fidelity time sequence fluorescent DNA bar code comprises a coding chain and a reading chain, the coding chain is formed by connecting three sections of coding areas and four sections of positioning areas in series; the reading chain comprises a reading chain 1, a reading chain 2 and a reading chain 3; after the reading chain 1 is specifically combined with the coding chain, a signal carried on the reading chain 1 is generated; the reading chain 2 replaces and releases the reading chain 1 combined on the bar code through a toehold-mediated chain replacement reaction, and generates a signal carried by the reading chain 2; the reading chain 3 replaces and releases the reading chain 2 combined on the bar code through a toehold-mediated chain replacement reaction, and generates a signal carried by the reading chain 3; therefore, a time sequence change signal which can be detected is generated and is used for identifying the target molecule. The DNA bar code can exponentially expand the coding capability of the DNA bar code, and in-situ multiple imaging and analysis of various proteins, nucleic acids or polysaccharides can be easily realized.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to a high-fidelity time-series fluorescent DNA barcode and its applications. Background Technology

[0002] Multiplex molecular imaging is a key technology in systems biology and disease diagnosis, allowing for the simultaneous mapping of the spatial distribution of multiple biomolecules in a single cell or tissue sample. However, traditional fluorescence-based imaging methods are severely limited by the spectral overlap of fluorescent dyes, typically making it difficult to detect more than five targets simultaneously.

[0003] To overcome this limitation, various strategies have been developed in the field. Among them, DNA-encoded strategies have attracted much attention due to their high programmability.

[0004] One approach is to use DNA nanotechnology to construct complex spatial barcodes, but this process is usually complex and costly.

[0005] Another type is the time-coding strategy, which encodes the target's identity as a time-series signal, greatly improving multiplexing capabilities. However, existing time-coding techniques generally suffer from the following drawbacks: 1. Relying on complex multi-stranded DNA nanostructures as coding units increases the risk of non-specific interactions and incomplete reactions.

[0006] 2. It lacks an inherent error correction mechanism and cannot actively correct any non-specific bindings that may occur, thus affecting the fidelity and accuracy of the final decoding.

[0007] Therefore, developing a novel multi-image platform with a simple structure and high-fidelity error correction capability is a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a high-fidelity time-series fluorescent DNA barcode and its application. The high-fidelity time-series fluorescent DNA barcode has a simple structure, inherent active error correction capability, and is a high-fidelity and highly scalable multiplex molecular imaging method, system and kit.

[0009] To address the aforementioned technical problems, this invention provides a high-fidelity time-series fluorescent DNA barcode, which includes a coding strand and a reading strand. The coding chain is composed of three coding regions and four positioning regions connected in the order of positioning region 1-coding region 1-positioning region 2-coding region 2-positioning region 3-coding region 3-positioning region 4; The coding region 1 is one of A, B, and C; the coding region 2 is one of A, B, and C; the coding region 3 is one of A, B, and C; The gene sequence of A is GCAT; the gene sequence of B is AAGG; and the gene sequence of C is GTTG. The gene sequence of the location region 1 is CTCGAAG, the gene sequence of the location region 2 is GTCGCAC, the gene sequence of the location region 3 is GACTGAG, and the gene sequence of the location region 4 is TACG. The read chain includes a read chain 1 specifically bound to the coding region 1, a read chain 2 specifically bound to the coding region 2, and a read chain 3 specifically bound to the coding region 3. First round: After the read chain 1 specifically combines with the coding region 1, the signal carried on the read chain 1 is generated; Second round: Read chain 2 replaces and releases read chain 1, which is attached to the encoding chain, through a chain substitution reaction mediated by toehold, generating a signal carried on read chain 2; Third round: Read chain 3 replaces and releases read chain 2, which is attached to the encoded chain, through a chain substitution reaction mediated by toehold, generating a signal carried on read chain 3; In each round of operation, the read chain replaces and releases the read chain that was bound to the coding chain in the previous round through a toehold-mediated chain displacement reaction, thereby generating a detectable temporal change signal at the location of the analyte; the target molecule is identified based on the time sequence of the signal.

[0010] In the aforementioned high-fidelity time-series fluorescent DNA barcode, further, the readout strand 1 is one of R1A, R1B, and R1C; the readout strand 2 is one or more of R2AA, R2AB, R2AC, R2BA, R2BB, R2BC, R2CA, R2CB, and R2CC; and the readout strand 3 is one or more of R3AA, R3AB, R3AC, R3BA, R3BB, R3BC, R3CA, R3ACB, and R3CC. The gene sequence of R1A is shown in SEQ ID NO.1, the gene sequence of R1B is shown in SEQ ID NO.2, and the gene sequence of R1C is shown in SEQ ID NO.3; The gene sequence of R2AA is shown in SEQ ID NO.4, the gene sequence of R2AB is shown in SEQ ID NO.5, the gene sequence of R2AC is shown in SEQ ID NO.6, the gene sequence of R2BA is shown in SEQ ID NO.7, the gene sequence of R2BB is shown in SEQ ID NO.8, the gene sequence of R2BC is shown in SEQ ID NO.9, the gene sequence of R2CA is shown in SEQ ID NO.10, the gene sequence of R2CB is shown in SEQ ID NO.11, and the gene sequence of R2CC is shown in SEQ ID NO.12. The gene sequence of R3AA is shown in SEQ ID NO.13, the gene sequence of R3AB is shown in SEQ ID NO.14, the gene sequence of R3AC is shown in SEQ ID NO.15, the gene sequence of R3BA is shown in SEQ ID NO.16, the gene sequence of R3BB is shown in SEQ ID NO.17, the gene sequence of R3BC is shown in SEQ ID NO.18, the gene sequence of R3CA is shown in SEQ ID NO.19, the gene sequence of R3ACB is shown in SEQ ID NO.20, and the gene sequence of R3CC is shown in SEQ ID NO.21.

[0011] Furthermore, in the aforementioned high-fidelity time-series fluorescent DNA barcode, the read strand 1, read strand 2, and read strand 3 each carry different fluorescent groups.

[0012] Furthermore, the high-fidelity time-series fluorescent DNA barcode described above also includes an auxiliary strand, which can bind to the probe of the high-fidelity time-series fluorescent DNA barcode and activate an efficient strand substitution reaction between the mismatched read strand and the barcode. The auxiliary chain is one of Helper A, Helper B, and Helper C; The gene sequence of Helper A is shown in SEQ ID NO.22; The gene sequence of Helper B is shown in SEQ ID NO.23; The gene sequence of Helper C is shown in SEQ ID NO.24.

[0013] Furthermore, the high-fidelity time-series fluorescent DNA barcode described above also includes a wash-free module; The no-wash module includes a quenching chain E1 capable of hybridizing read chain 2 and read chain 1, and a quenching chain E2 capable of hybridizing read chain 3 and read chain 2.

[0014] The gene sequence of the quenching chain E1 is shown in SEQ ID NO.25; the gene sequence of the quenching chain E2 is shown in SEQ ID NO.26.

[0015] The aforementioned high-fidelity time-series fluorescent DNA barcode may further include a secondary antibody conjugate or a phalloidin conjugate linked to the coding strand.

[0016] The aforementioned high-fidelity time-series fluorescent DNA barcode further employs a guanidine isothiocyanate-sulfo-SMCC (Sulfo-SMCC) cross-linking agent. The NHS ester at one end of the cross-linking agent reacts with the primary amine or amino-modified phalloidin of the secondary antibody, while the maleimide at the other end reacts with the thiol group of the coding strand after DTT reduction to form a stable covalent bond.

[0017] Based on a general technical concept, the present invention provides an application of the high-fidelity time-series fluorescent DNA barcode in the preparation of multiplex analytical instruments.

[0018] Based on a general technical concept, the present invention provides an application of the high-fidelity time-series fluorescent DNA barcode in the preparation of a detection kit for in situ multiplex imaging and analysis of various proteins, nucleic acids or polysaccharides.

[0019] Compared with the prior art, the advantages of the present invention are as follows: (1) This invention provides a high-fidelity time-series fluorescent DNA barcode that uses a single-stranded DNA backbone as the carrier of the coding region. Considering the maximization of thermodynamic advantages, the specificity of the read strand hybridization is ensured by using common positioning regions. At the same time, this structure integrates an inherent kinetic correction mechanism, which can actively suppress and reverse non-specific reactions to ensure the accuracy of the encoding. It is easy to design and synthesize, and has strong stability and anti-interference ability.

[0020] (2) The present invention provides a high-fidelity time-series fluorescent DNA barcode. The sequence imaging kinetics is based on the DNA sequence strand displacement reaction, which has a fast reaction rate and good stability.

[0021] (3) This invention provides a high-fidelity time-series fluorescent DNA barcode. Through structured and modular design, its coding capability can be expanded exponentially. Taking M fluorescent groups and N coding regions as an example, MN coding combinations can be generated, easily achieving high throughput.

[0022] (4) This invention provides a high-fidelity time-series fluorescent DNA barcode with good scalability and multifunctionality. It can integrate plug-and-play modules with different functions (such as auxiliary strand modules and no-wash modules) without changing the original core structure and sequence, thus optimizing cost and process.

[0023] (5) This invention provides an application of high-fidelity time-series fluorescent DNA barcodes, successfully applying the HiFi-TFDB system to in situ cell analysis, achieving high-fidelity, simultaneous multiplex imaging of various endogenous proteins and cell structures. This invention can be directly applied to the core scenario of biological research—cell imaging; in the crowded intracellular environment, different time-series coding channels can work in parallel without interfering with each other; the DNA barcode of this invention can be easily coupled with various molecular probes (antibodies, phalloidin, etc.), possessing broad application expansion potential. Attached Figure Description

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0025] Figure 1 This is a schematic diagram illustrating the design principle of the HiFi-TFDB platform and its use for multiple imaging in an embodiment of the present invention.

[0026] Figure 2 This provides the intrinsic dynamic correction principle and experimental verification of the HiFi-TFDB platform in Experiment 1 of this invention.

[0027] Figure 3 This is a schematic diagram and experimental verification of the auxiliary chain module of the HiFi-TFDB platform in Experiment 2 of this invention.

[0028] Figure 4 This is a schematic diagram and experimental verification of the no-wash module of the HiFi-TFDB platform in Experiment 3 of this invention.

[0029] Figure 5 This is a schematic diagram and experimental verification of the HiFi-TFDB platform used for temporal imaging of nine types of coded microspheres in Embodiment 4 of the present invention.

[0030] Figure 6 This is a schematic diagram and experimental verification of the HiFi-TFDB platform used for protein multiple temporal imaging in Embodiment 5 of the present invention. Detailed Implementation

[0031] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention. All materials and instruments used in the following embodiments are commercially available.

[0032] Cellular secretome source: The human cervical cancer cells HeLa used in this experiment were purchased from the Cell Bank of the Chinese Academy of Sciences. DNA sequence source: All DNA sequences used in this experiment were synthesized and purified by Shanghai Sangon Biotech Co., Ltd. Polystyrene microspheres source: Shaanxi Xingbei Aike Co., Ltd.

[0033] The nucleotide sequence information used in this invention is shown in Table 1.

[0034] Table 1: Nucleotide sequence information used in this invention

[0035] In Table 1, the bolded part is A, the underlined part is B, and the italicized part is C.

[0036] The sequence of the encoding chain Bar is: CTCGAAG XXXX GTCGCAC XXXX GACTGAG XXXX TACG, where XXXX represents one of A, B, and C, where A is GCAT, B is AAGG, and C is GTTG.

[0037] The sequence R1-X of the first-round readout strand is: CGTA XXXX CTCAGTC; the sequence R2-X of the second-round readout strand is: XXXX CTCAGTC XXXX GTGCGAC; the sequence R3-X of the third-round readout strand is: CTCAGTC XXXX GTGCGAC XXXXCTTCGAG; in the sequences, XXXX represents one of A*, B*, and C*, where A* is ATGC, B* is CCTT, and C* is CAAC. If the readout strand carries a fluorescent group, the gene sequence is: R1-X: CGTA XXXX CTCAGTC T-fluorescent group; R2-XX:XXXX CTCAGTC XXXX GTGCGAC T-fluorescent group; R3-XX: CTCAGTC XXXX GTGCGAC XXXX CTTCGAG T-fluorescent group.

[0038] See Figure 1The coding strand consists of three 4-base address domains and four location domains connected in series. The first location domain (L1) is 4 bases long, while the other three (L2, L3, and L4) are 7 bases long. Specifically, the sequence is: Location Domain 1 - Coding Domain 1 - Location Domain 2 - Coding Domain 2 - Location Domain 3 - Coding Domain 3 - Location Domain 4. Each coding domain is divided into three segments, and different sequences in each segment determine different fluorescent groups. Taking three fluorescent groups—Cy3, Cy5, and FAM—as examples, their corresponding coding domain sequences are represented by A, B, and C, respectively. Based on different combinations, 27 different coding strand libraries can be generated.

[0039] The location region is divided into four segments, whose positions and sequences on the coding chain are fixed and constitute the common sequence of the entire coding chain library.

[0040] Different barcode chains are coupled to different target molecules (e.g., via antibodies), and the imaging principle is as follows: First round: The readout chain 1 with fluorescent marker 1 specifically binds to the localization region 1-coding region 1-localization region 2, at which point the target generates fluorescent signal 1.

[0041] In the second round, the read chain 2 with fluorescent marker 2 specifically binds to the coding region 1-location region 2-coding region 2-location region 3. The read chain 2 replaces and releases the read chain 1 that was bound to the barcode in the previous round through a toehold-mediated chain displacement reaction, thereby generating a detectable time-varying signal at the location of the analyte: a new fluorescent signal 2.

[0042] Third round: The read chain 3 with fluorescent marker 3 specifically binds to the positioning region 2-coding region 2-positioning region 3-coding region 3-positioning region 4. The read chain 3 replaces and releases the read chain 2 that was bound to the barcode in the previous round through a toehold-mediated chain displacement reaction, thereby generating a detectable temporal change signal at the location of the analyte: a new fluorescent signal 3.

[0043] Repeating this operation will cause the fluorescence signal on the target molecule to exhibit a predetermined sequence change over time (e.g., red → green → yellow). By recording the fluorescence color sequence at each target location, the identity of the target can be accurately identified.

[0044] Example 1 A high-fidelity temporal fluorescent DNA barcode (HiFi-TFDB). The high-fidelity temporal fluorescent DNA barcode includes a library of 27 code strands (see Table 1) and a library of 21 fluorescently modified read strands (see Table 1).

[0045] Based on 27 coding chain libraries and 21 read chain libraries, we selected nine representative coding chains and their corresponding read chains, biotinylated them, and modified them on the surface of streptavidin-coated polystyrene microspheres for the display of multiple temporal imaging of the nine coding chains. The biotinylation coding chains are BarABC-Biotin, BarCBA-Biotin, BarBAC-Biotin, BarBBA-Biotin, BarBCC-Biotin, BarCBC-Biotin, BarCCA-Biotin, BarCCC-Biotin, and BarBBB-Biotin, where Biotin represents biotin. A represents the sequence determining Cy3, B represents the sequence determining Cy5, and C represents the sequence determining FAM.

[0046] The first round of read chains 1 consists of R1A-Cy3, R1B-Cy5, and R1C-FAM; the second round of read chains 2 consists of R2AB-Cy5, R2BA-Cy3, R2BB-Cy5, R2BC-FAM, R2CB-Cy5, and R2CC-FAM; the third round of read chains 3 consists of R3BC-FAM, R3AC-FAM, R3BA-Cy3, R3BB-Cy5, R3CC-FAM, and R3CA-Cy3.

[0047] When the encoding chain is BarABC, a fluorescence signal of Cy3 is generated when the read chain 1 is R1A-Cy3; no fluorescence signal is generated when the read chain 1 is R1B-Cy5 or R1C-FAM.

[0048] When read chain 2 is R2AB-Cy5, it replaces and releases read chain 1 that is already bound to the coding chain through a toehold-mediated chain substitution reaction, generating a fluorescent signal of Cy5. Other read chains 2 do not generate fluorescent signals.

[0049] When read chain 3 is R3BC-FAM, read chain 2, which is bound to the coding chain, is replaced and released through a toehold-mediated chain substitution reaction, generating a FAM fluorescence signal. Other read chains 3 do not generate fluorescence signals.

[0050] Experiment 1: The underlying dynamic correction mechanism of the high-fidelity time-series fluorescent DNA barcode in Example 1 was investigated.

[0051] To monitor kinetics, the 3' end of BarABC was labeled with the FAM fluorescent group, and the 5' end of R1A and R2AB was labeled with the quenching group BHQ1. The specific experimental procedure is as follows: 1.1 Preparation of the initial reaction complex: The fluorescently labeled probe BarABC-FAM and the quencher-labeled probe R1A-BHQ1 were added to a buffer solution (components: 100 mM NaCl, 20 mM Tris-HCl, pH 8.2) at a molar ratio of 1:1.2 and mixed thoroughly. The mixture was then annealed to form the initial complex BarABC:R1A. The annealing program was set as follows: first, the temperature was held at 95°C for 5 minutes, then annealed at a cooling rate of 1.2°C / min until it reached 25°C, and then rapidly cooled to 4°C. In this initial complex, the FAM fluorescent group and the BHQ1 quencher group are in close contact, resulting in a quenched fluorescence signal.

[0052] 1.2. Gel electrophoresis verification of the kinetic correction mechanism: A 15% non-denaturing polyacrylamide gel was prepared (1 mL of 5×TBE solution, 3.75 mL of 40% Arc / Bis gel preparation solution, 0.1 mL of 10% ammonium persulfate solution, and 5.15 mL of ultrapure water were added sequentially to a 10 mL centrifuge tube, vortexed until homogeneous, and then 4 μL of TEMED reagent was added and thoroughly mixed to obtain the 15% non-denaturing polyacrylamide gel). Multiple parallel reaction tubes were set up, each containing an initial BarABC:R1A complex at a final concentration of 1 µM.

[0053] Control group: Mismatch probe R2BB and mismatch probe R2CB with a final concentration of 1 µM were added to the reaction tubes respectively.

[0054] Calibration group: The perfectly matched probe R2AB and multiple mismatched probes were added to the reaction tube at the same time, and two concentration ratio gradients were set. In one group, the total concentration ratio of R2AB to all mismatched probes was 1:1, and in the other group it was 2:1.

[0055] All reaction tubes were placed at a constant temperature of 25°C for 1 hour. After the reaction, each reaction sample was subjected to 15% non-denaturing polyacrylamide gel electrophoresis (PAGE). Electrophoretic band images were acquired using a gel imaging system to analyze the formation of nonspecific products in each reaction group and the inhibitory effect of the kinetic correction mechanism on nonspecific products.

[0056] 1.3 Implementation and Validation of the Kinetic Correction Mechanism: A perfectly matched probe R2AB and a mismatched probe R2BB, each with a final concentration of 400 nM, were simultaneously added to the initial BarABC:R1A complex at a final concentration of 200 nM and thoroughly mixed to obtain the reaction system. The reaction system was monitored in real-time using a fluorescence spectrophotometer at a constant temperature of 25℃, and the dynamic changes in fluorescence intensity were continuously recorded.

[0057] Figure 2 The results are for kinetic verification. Figure 'a' illustrates the kinetic verification principle: When the mismatched probe R2BB preferentially reacts with the initial BarABC:R1A complex, it displaces R1A-BHQ1 bound to BarABC, causing the FAM fluorophore to leave the effective range of the BHQ1 quencher group, resulting in an increase in the system's fluorescence signal. Subsequently, the perfectly matched probe R2AB specifically binds to BarABC, displacing the bound R1A-BHQ1. At this point, because R2AB carries the quencher group, the system's fluorescence signal remains stable. Further, R2AB displaces the bound mismatched probe R2BB, forming a stable BarABC:R2AB final product, causing the system's fluorescence signal to decrease. Finally, a complete kinetic correction curve of "fluorescence increase-decrease" can be collected, thus verifying the effectiveness of the kinetic correction mechanism.

[0058] Figure b shows the PAGE detection results. When only the mismatched probe (MM) is present in the reaction system, the non-specific product band can be clearly observed, indicating that the mismatched probe easily reacts non-specifically with the initial complex BarABC:R1A and generates the wrong product. When the perfectly matched probe (PM, i.e., R2AB) is added to the system and the total concentration ratio of PM to MM is 1:1, the intensity of the non-specific product band is significantly reduced, and the target product band becomes the dominant band; when the total concentration ratio of PM to MM is increased to 2:1, the non-specific product band almost completely disappears. This result intuitively confirms that the presence of the perfectly matched probe (R2AB) can actively correct or inhibit the non-specific reaction caused by the mismatched probe, and its inhibitory effect increases with the increase of its own concentration ratio, confirming the effectiveness of the kinetic correction mechanism.

[0059] Figure c shows that in the reaction system where the perfectly matched probe (PM) and the mismatched probe (MM) coexist, the fluorescence kinetic curve exhibits a typical "rise followed by fall" characteristic. This is direct evidence of the occurrence of the kinetic correction mechanism. The specific reaction process is as follows: First, the mismatched probe R2BB reacts faster kinetically, preferentially binding to the initial complex BarABC:R1A and displacing R1A-BHQ1, thus removing the FAM fluorescent group from the quenching effect, resulting in an increase in the fluorescence signal of the system. During this stage, an unstable transient error product is generated. Second, the thermodynamically more stable perfectly matched probe R2AB (R2AB-BHQ1) gradually replaces the weakly bound mismatched probe, reforming the stable final product BarABC:R2AB carrying the quenching group, causing the fluorescence signal of the system to decrease and tend to stabilize.

[0060] Figure d shows that the peak height of the transient fluorescence signal is positively correlated with the initial concentration of the mismatch probe (MM): the higher the concentration of the mismatch probe, the more transient error products are formed by its binding with the initial complex, the greater the total amount of FAM fluorescent groups detached from quenching, and the higher the corresponding fluorescence peak. This result further corroborates the working process of the kinetic correction mechanism and shows that the amount of transient error products generated in the system can be intuitively reflected by the fluorescence peak, providing a basis for subsequent quantitative detection applications based on this mechanism.

[0061] Example 2 A set of HiFi-TFDB sequences for verifying the kinetic correction of the coding system includes a coding strand modified with FAM fluorescence, a perfectly matched readout strand (PM) with BHQ1 quenching group, a mismatched readout strand (a readout strand that can specifically identify coding region 3 but cannot specifically identify coding region 2), and an auxiliary strand (one of Helper-A, Helper-B, and Helper-C).

[0062] The gene sequence of the helper chain Helper-X is: GTCGCAC XXXX GACTGAG, where XXXX is one of A, B, and C, where A is GCAT, B is AAGG, and C is GTTG.

[0063] For example, for the coding chains Bar-ABC and Bar-ACC, the first-round readout chain 1 is R1A-Cy5; the second-round readout chains 2 are R2AB-Cy3 and R2AC-Cy3. If the third-round fluorescent readout chain is chosen as R3-CC, for Bar-ACC, R3-CC is a perfectly matched chain (PM), which can directly and efficiently bind and generate a fluorescent signal; for Bar-ABC, R3-CC is a mismatched readout chain (MM), and without an auxiliary chain, the reaction efficiency is extremely low, making it difficult to detect the signal.

[0064] In the above process, after adding the corresponding helper chain Helper-B, Helper-B binds to the corresponding position in coding region 2, where R3-CC cannot bind, through a toehold-mediated chain substitution reaction. This replaces and releases the R3-CC originally bound to Bar-ABC, ultimately achieving simultaneous imaging and detection of both Bar-ABC and Bar-ACC coding chains without the need for an additional third-round readout chain. This creates an efficient reaction path for mismatched readout chains through allosteric effects or by providing a new toehold for chain substitution.

[0065] Experiment 2: Verify the feasibility of the HiFi-TFDB platform auxiliary chain module.

[0066] (1) Microsphere sample preparation: A mixed microsphere population containing three different coding chains (BarBAC, BarABC, BarCCC) was prepared to complete the second chain replacement reaction, so that the three microspheres have unique fluorescence signals, and the microspheres can be distinguished and identified in the field of view.

[0067] (2) Functional verification of the auxiliary chain module (imaging): A readout chain mixture containing mismatched chains R3CC and R3BC was added to the above-mentioned mixed microsphere system. Since R3CC and R3BC are mismatched with the BarBAC coding chain, theoretically no reaction or only a very low-efficiency reaction should occur. After incubation at room temperature, imaging was performed using a confocal microscope. After the first imaging was completed, Helper AC auxiliary chain was added to the same reaction system, and imaging was performed again after incubation at room temperature to observe the changes in the fluorescence signal of the system.

[0068] (3) Functional verification of the auxiliary chain module (real-time kinetics): To monitor the process of the auxiliary chain regulating the reaction in real time, the relevant probes were labeled: BarBAC was labeled with the FAM fluorescent group, and R2BA was labeled with the BHQ1 quencher group. The reaction system was constructed: containing a BarBAC:R2BA complex with a final concentration of 200 nM and R3CC with a final concentration of 400 nM, and the changes in the fluorescence signal of the system were monitored; after the reaction proceeded for a period of time, a Helper AC auxiliary chain with a final concentration of 400 nM was added to the system, and the dynamic changes in the fluorescence intensity of the system were continuously monitored.

[0069] Figure 3This diagram illustrates the auxiliary chain module of the HiFi-TFDB platform and provides verification results. Figure 'a' represents the schematic diagram of the auxiliary chain module. This embodiment aims to demonstrate that the HiFi-TFDB system described in this invention possesses excellent modular scalability. By introducing a "plug-and-play" auxiliary chain module, the system's cost-effectiveness and application flexibility can be significantly improved. The core objective of this module is to use a smaller, degenerate fluorescent probe library to decode a barcode library much larger than itself, thereby drastically reducing the synthesis cost of fluorescent probes. The principle is as follows: a mismatched metabolite (MM) with extremely low reaction efficiency will have its chain substitution reaction efficiency activated and improved to near-perfectly matched probe PM when a corresponding "helper strand" is present. Specifically, the helper strand first binds to a specific site on the coding strand, creating an efficient reaction path for the mismatched MM through allosteric effects or by providing a new chain substitution toehold. This reaction is strictly dependent on the presence of the helper strand.

[0070] Figure b shows the imaging results: when only mismatched chains R3CC and R3BC were added (top right of figure b), the two control microspheres, BarABC and BarCCC, showed corresponding fluorescence signal changes, while the BarBAC microsphere did not produce the green fluorescence signal corresponding to R3CC; after adding the Helper AC auxiliary chain (bottom left of figure b), the BarBAC-encoded microsphere was specifically illuminated green. This result clearly confirms that the reaction between the mismatched chain and the BarBAC-encoded chain strictly depends on the presence of the specific Helper AC auxiliary chain, and the auxiliary chain can effectively activate the targeting reaction. The kinetic curves show that before adding the Helper AC auxiliary chain, the fluorescence signal of the system remained basically at the background level, with no obvious reaction signal; after adding the Helper AC auxiliary chain, the fluorescence signal of the system increased rapidly and sharply, indicating that the auxiliary chain can efficiently activate the chain substitution reaction.

[0071] This embodiment successfully verifies the feasibility of the HiFi-TFDB platform's auxiliary chain module. This module precisely controls the reactivity of mismatched probes by adjusting the presence or absence of an auxiliary chain, allowing a limited set of degenerate fluorescent probes to decode a vast and diverse barcode library by combining different auxiliary chains. This module significantly reduces system application costs while enhancing design flexibility, highlighting the application potential and advantages of the system of this invention.

[0072] Example 3 A high-fidelity time-series fluorescent DNA barcode is developed based on Example 2, with the addition of a wash-free module. This wash-free module simplifies the experimental process in multi-round imaging, eliminating cumbersome physical washing operations between steps.

[0073] The no-wash module includes a quenching strand E1 capable of hybridizing read strand 2 and read strand 1, and a quenching strand E2 capable of hybridizing read strand 3 and read strand 2; in this embodiment, the quenching strands used are quenching strands E1A and E2AB, and their gene sequences are shown in Table 1.

[0074] Experiment 3: Verify the feasibility of the HiFi-TFDB platform's no-wash module.

[0075] (1) PAGE verification of module feasibility: Polyacrylamide gel electrophoresis (PAGE) was used to analyze the state of each component in the wash-free module before and after the reaction, including the initial complex, the pre-quenched readout probe complex, and the final product after the reaction, to verify whether the chain substitution reaction and the quenching capture reaction occurred as expected. The final concentration of each reaction component added in the experiment was set to 1 µM.

[0076] (2) Real-time kinetic verification of module function: The reaction process was monitored in real time by a fluorescence spectrophotometer. Taking the first step reaction as an example, the experimental system was constructed: In the BarABC:R1A-Cy3 complex system with a final concentration of 200 nM, the pre-quenching complex R2AB-Cy5:E1A-BHQ2 with a final concentration of 400 nM was added. After thorough mixing, the changes of two fluorescence signals were monitored in real time: one was the attenuation of Cy3 fluorescence signal (reflecting the process of R1A being replaced and quenched by E1A), and the other was the enhancement of Cy5 fluorescence signal (reflecting the process of R2AB binding to the coding strand).

[0077] Figure 4 This diagram illustrates the no-wash module of the HiFi-TFDB platform in this embodiment and shows the verification results. As shown in Figure a, the core design of this no-wash module is a dual quenching mechanism, as detailed below: (1) Transfer quenching: The read chain 2 (such as R2AB) entering the system exists in the form of a pre-quenched double-stranded complex (R2AB:E1), where E1 is a quenching chain with a quenching group, and the complex itself is non-fluorescent; (2) Dual quenching: When R2AB dissociates from the complex and replaces the read chain 1 (such as R1A) bound to the coding chain in the previous round, the released E1 chain immediately captures and quenches the free R1A in the solution that has been replaced. Through this "bottom-up" dual quenching mechanism, the background fluorescence generated by the excess read chain and the replaced read chain in the system can be effectively eliminated, achieving "wash-free" operation.

[0078] As shown in Figure b, the gel electrophoresis results clearly show that the band positions of each intermediate and the final product are completely consistent with the theoretical design, proving that the chain displacement reaction and quenching capture reaction in the wash-free module can occur efficiently and accurately as expected.

[0079] As shown in Figure c, the kinetic curve clearly illustrates the signal transition process. After the addition of the second round of pre-quenching complex, the Cy3 fluorescence signal from the first round rapidly decayed, while the Cy5 fluorescence signal from the second round steadily increased. Ultimately, the fluorescence state of the system smoothly transitioned from Cy3-dominated to Cy5-dominated. This result indicates that both the displaced probe and excess probe in the solution were effectively quenched, successfully achieving signal switching under wash-free conditions.

[0080] This embodiment successfully demonstrates the feasibility of the wash-free functional module. Through an ingenious "double quenching" design, this module effectively solves the problem of background fluorescence interference in multi-round reactions, thereby eliminating the need for physical cleaning steps. The successful construction of this module greatly simplifies experimental operations and facilitates the automation of the technology of this invention.

[0081] Example 4 Application of fluorescent DNA barcodes from Example 1 in time-series imaging of nine types of encoded microspheres.

[0082] Materials used in this embodiment: streptavidin-coated polystyrene microspheres (5 µm in diameter), reaction buffer (components: 100 mM NaCl, 20 mM Tris-HCl, pH 8.2), and washing buffer (0.05% Tween-20 added to the reaction buffer).

[0083] Application method: (1) Preparation of 9-fold encoded microsphere mixture: The 9 biotin-modified coding chains in Example 1 were incubated with streptavidin-coated microspheres at 4°C overnight to prepare 9 microsphere populations carrying unique barcodes; each microsphere was washed three times with washing buffer to remove unbound free coding chains; the 9 coding microspheres were mixed in equal amounts to construct a heterogeneous microsphere population containing multiple coding information.

[0084] (2) Iterative imaging process of temporal decoding: First round: Add the first round readout strand mixture containing R1A-Cy3, R1B-Cy5, and R1C-FAM to the mixed microsphere sample, incubate at room temperature for 20 minutes to allow the readout strand to hybridize with the first site of the coding strand; after washing, use a confocal microscope to image in the three channels of Cy3, Cy5, and FAM to record the first round fluorescence signal of each microsphere.

[0085] Second round: Add a mixture containing all the second-round readout strands to the same microsphere sample and incubate for 20 minutes. At this time, the second-round readout strand (R2) replaces the bound first-round readout strand (R1) through a chain displacement reaction. After washing, perform a second three-channel imaging to record the second-round fluorescence signal of each microsphere.

[0086] Third round: Repeat the above iterative steps, add the third round read chain mixture, complete the third three-channel imaging, and record the final fluorescence signal of each microsphere.

[0087] (3) Data analysis and decoding: The image data of each microsphere in the three rounds of imaging are systematically analyzed. The decoding process adopts a qualitative identification method: by judging whether there is a signal in the fluorescence channel in each round of imaging, the color signal corresponding to that round is determined; the color signals of the three rounds are combined in sequence to obtain the unique time sequence barcode carried by each microsphere (for example, “Cy3→Cy5→FAM” corresponds to BarABC); based on the barcode information obtained by decoding, each microsphere is assigned a unique pseudo-color to generate the final decoded spectrum.

[0088] Figure 5 This is a schematic diagram and verification results of the HiFi-TFDB platform used for temporal imaging of nine types of coded microspheres. Figure 'a' is a schematic diagram that verifies whether the system can decode multiple targets simultaneously and orthogonally without significant crosstalk by constructing a hybrid microsphere system containing nine different temporal barcodes.

[0089] As shown in Figure b, the confocal microscope image visually presents the complete decoding process: after the first readout chain is added, the mixed microspheres exhibit three initial colors: yellow (Cy3), red (Cy5), and green (FAM); with the sequential addition of the second and third readout chains, the color distribution of the microsphere population undergoes the expected programmed dynamic change.

[0090] Figure c shows in detail the color change trajectories of nine representative microspheres in three rounds of imaging: for example, the microsphere marked "Barcode 1" strictly follows the order of "yellow (Cy3) → red (Cy5) → green (FAM)" in fluorescence signal, which is completely consistent with the preset code of BarABC; the other eight microspheres also show unique and crosstalk-free color change sequences.

[0091] Figure d shows that in the final generated decoding map, each pseudo-color corresponds to a successfully decoded barcode; the random distribution of 9 different colored microspheres proves that the system can successfully and with high throughput identify and distinguish all 9 targets from a complex mixed system, realizing the expected multi-analysis capability.

[0092] This embodiment fully demonstrates the core capabilities of the HiFi-TFDB system as a multi-analysis platform. The experimental results are specifically reflected in: (1) High throughput: The platform can simultaneously analyze and decode multiple (9 in this embodiment) different targets in the same sample; (2) High orthogonality: There is no obvious crosstalk between the 9 time-series codes, and each barcode can be accurately identified by the corresponding reading chain sequence, resulting in high decoding accuracy; (3) Decoding robustness: Decoding depends on the presence or absence of qualitative signals rather than precise fluorescence ratio calculation, which makes the system highly resistant to experimental noise such as photobleaching and probe density unevenness.

[0093] Example 5 A fluorescent DNA barcode, with an antibody and phalloidin conjugated to the coding strand of Example 1.

[0094] Specifically, a guanidine isothiocyanate-sulfonyl-SMCC (Sulfo-SMCC) crosslinking agent is used. The NHS ester at one end of the crosslinking agent reacts with the primary amine group of the secondary antibody, and the maleimide at the other end reacts with the thiol group of the coding chain after DTT reduction to form a stable covalent bond, thus obtaining the coding chain-secondary antibody conjugate. By using the same chemical method to link the coding chain with an amino-modified phalloidin, a coding chain-phalloidin conjugate was obtained.

[0095] This embodiment describes the application of fluorescent DNA barcodes in multiplex immunofluorescence imaging of endogenous proteins in cells. This embodiment aims to verify the practical application performance of the HiFi-TSD system described in this invention in real biological samples, that is, to perform simultaneous, in situ, high-fidelity multiplex imaging of multiple endogenous proteins in fixed mammalian cells.

[0096] Materials used in this embodiment: cell culture medium, 4% paraformaldehyde (PFA), 0.1% Triton X-100, bovine serum albumin (BSA), salmon sperm DNA, and guanidine isothiocyanate-sulfo-SMCC (Sulfo-SMCC) crosslinking agent.

[0097] Application method: (1) Preparation of coding chain-antibody / phalloidin conjugate: The conjugate was constructed using Sulfo-SMCC crosslinking agent: the NHS ester at one end of the crosslinking agent reacted with the primary amino group of the secondary antibody, and the maleimide at the other end reacted with the thiol group of the coding chain after DTT reduction to form a stable covalent bond, thus obtaining the coding chain-secondary antibody conjugate; the coding chain was then linked to an amino-modified phalloidin using the same chemical method to obtain the coding chain-phalloidin conjugate. The reaction product was purified by ultrafiltration to remove unreacted DNA.

[0098] (2) Cell sample preparation and multi-target labeling: HeLa cells were cultured in confocal dishes. After the cells adhered, they were fixed with 4% PFA for 10 minutes and then washed with PBS buffer. 0.1% Triton X-100 was added to permeate the cell membrane for 5 minutes. Blocking solution containing 5% BSA and 1 mg / mL salmon sperm DNA was used and the cells were blocked at room temperature for 1 hour. Multiple primary antibodies (such as anti-Golgin-97 antibody, anti-CD44 antibody, and anti-Lamin B1 antibody) were mixed and incubated with the cells at 4°C overnight. After washing, a mixture of secondary antibody conjugates corresponding to the primary antibody and each carrying a different coding strand (or an additional coding strand-phalloidin conjugate) was added and incubated at room temperature for 2 hours.

[0099] (3) Timing decoding and imaging: After the target protein labeling is completed, the same sample is subjected to three rounds of "hybridization-washing-imaging" cycle operation: R1, R2 and R3 reading chain mixtures are added in each round and incubated at room temperature for 30 minutes; after each round of incubation, the sample is washed and fluorescent images are acquired in the Cy3, Cy5 and FAM channels using a confocal microscope.

[0100] Figure 6 This diagram illustrates the application of the HiFi-TFDB platform for protein multiplex temporal imaging in this embodiment of the disclosure, along with verification results. Figure a shows a schematic diagram of the HiFi-TFDB platform used for protein multiplex temporal imaging.

[0101] As shown in Figure b, this invention successfully achieved simultaneous multiplex imaging of three endogenous proteins: Golgin-97, CD44, and Lamin B1. Through temporal decoding, the subcellular localization of the three proteins was clearly resolved: Golgin-97 is located in the Golgi apparatus, CD44 in the cell membrane, and Lamin B1 in the nuclear membrane. The fluorescence signal in the Golgi region strictly followed the preset CBA coding (green→red→yellow) change, the cell membrane region followed the ABC coding (yellow→red→green) change, and the nuclear membrane region followed the BAC coding (red→yellow→green) change. The three signaling pathways were spatially clearly defined and temporally completely orthogonal, with no obvious signal crosstalk observed, demonstrating the high fidelity of this system in complex cellular environments.

[0102] As shown in Figure c, this invention further demonstrates its ability to integrate different types of probes, successfully achieving simultaneous imaging of Golgin-97 (antibody-labeled), F-actin (phalloidin-labeled), and Lamin B1 (antibody-labeled). The results clearly show the precise localization of the three targets (Golgi apparatus, filamentous pseudopodia, and nuclear membrane), and all three targets exhibit unique temporal color changes completely consistent with their preset codes, without interference between them. These results demonstrate that the barcode system of this invention possesses good modularity and compatibility, and can be easily extended to other molecular recognition elements besides antibodies.

Claims

1. A high-fidelity time-series fluorescent DNA barcode, characterized in that, The high-fidelity time-series fluorescent DNA barcode includes a coding strand and a reading strand; The coding chain is composed of three coding regions and four positioning regions connected in the order of positioning region 1-coding region 1-positioning region 2-coding region 2-positioning region 3-coding region 3-positioning region 4; The coding region 1 is one of A, B, and C; the coding region 2 is one of A, B, and C; the coding region 3 is one of A, B, and C; The gene sequence of A is GCAT; the gene sequence of B is AAGG; and the gene sequence of C is GTTG. The gene sequence of the location region 1 is CTCGAAG, the gene sequence of the location region 2 is GTCGCAC, the gene sequence of the location region 3 is GACTGAG, and the gene sequence of the location region 4 is TACG. The read chain includes a read chain 1 specifically bound to the coding region 1, a read chain 2 specifically bound to the coding region 2, and a read chain 3 specifically bound to the coding region 3. First round: After the read chain 1 specifically combines with the coding region 1, the signal carried on the read chain 1 is generated; Second round: Read chain 2 replaces and releases read chain 1, which is attached to the encoding chain, through a chain substitution reaction mediated by toehold, generating a signal carried on read chain 2; Third round: Read chain 3 replaces and releases read chain 2, which is attached to the encoded chain, through a chain substitution reaction mediated by toehold, generating a signal carried on read chain 3; In each round of operation, the read chain replaces and releases the read chain that was bound to the coding chain in the previous round through a toehold-mediated chain displacement reaction, thereby generating a detectable temporal change signal at the location of the analyte; the target molecule is identified based on the time sequence of the signal.

2. The high-fidelity time-series fluorescent DNA barcode according to claim 1, characterized in that, The read chain 1 is one of R1A, R1B, and R1C; the read chain 2 is one or more of R2AA, R2AB, R2AC, R2BA, R2BB, R2BC, R2CA, R2CB, and R2CC; the read chain 3 is one or more of R3AA, R3AB, R3AC, R3BA, R3BB, R3BC, R3CA, R3ACB, and R3CC. The gene sequence of R1A is shown in SEQ ID NO.1, the gene sequence of R1B is shown in SEQ ID NO.2, and the gene sequence of R1C is shown in SEQ ID NO.3; The gene sequence of R2AA is shown in SEQ ID NO.4, the gene sequence of R2AB is shown in SEQ ID NO.5, the gene sequence of R2AC is shown in SEQ ID NO.6, the gene sequence of R2BA is shown in SEQ ID NO.7, the gene sequence of R2BB is shown in SEQ ID NO.8, the gene sequence of R2BC is shown in SEQ ID NO.9, the gene sequence of R2CA is shown in SEQ ID NO.10, the gene sequence of R2CB is shown in SEQ ID NO.11, and the gene sequence of R2CC is shown in SEQ ID NO.

12. The gene sequence of R3AA is shown in SEQ ID NO.13, the gene sequence of R3AB is shown in SEQ ID NO.14, the gene sequence of R3AC is shown in SEQ ID NO.15, the gene sequence of R3BA is shown in SEQ ID NO.16, the gene sequence of R3BB is shown in SEQ ID NO.17, the gene sequence of R3BC is shown in SEQ ID NO.18, the gene sequence of R3CA is shown in SEQ ID NO.19, the gene sequence of R3ACB is shown in SEQ ID NO.20, and the gene sequence of R3CC is shown in SEQ ID NO.

21.

3. The high-fidelity time-series fluorescent DNA barcode according to claim 2, characterized in that, The readout chain 1, readout chain 2 and readout chain 3 each carry different fluorescent groups.

4. The high-fidelity time-series fluorescent DNA barcode according to any one of claims 1 to 3, characterized in that, The high-fidelity time-series fluorescent DNA barcode also includes an auxiliary strand, which can activate a strand substitution reaction between the read strand that can specifically recognize coding region 3 but cannot specifically recognize coding region 2 and the coding strand. The auxiliary chain is one of Helper A, Helper B, and Helper C; The gene sequence of Helper A is shown in SEQ ID NO.22; The gene sequence of Helper B is shown in SEQ ID NO.23; The gene sequence of Helper C is shown in SEQ ID NO.

24.

5. The high-fidelity time-series fluorescent DNA barcode according to any one of claims 1 to 3, characterized in that, The high-fidelity time-series fluorescent DNA barcode also includes a wash-free module; The no-wash module includes a quenching chain E1 capable of hybridizing read chain 2 and read chain 1, and a quenching chain E2 capable of hybridizing read chain 3 and read chain 2; The quenching chain E1 and the quenching chain E2 are equipped with quenching groups.

6. The high-fidelity time-series fluorescent DNA barcode according to claim 5, characterized in that, The gene sequence of the quenching chain E1 is shown in SEQ ID NO.25; the gene sequence of the quenching chain E2 is shown in SEQ ID NO.

26.

7. The high-fidelity time-series fluorescent DNA barcode according to any one of claims 1 to 3, characterized in that, A secondary antibody conjugate or a phalloid peptide conjugate is attached to the coding strand.

8. The high-fidelity time-series fluorescent DNA barcode according to claim 7, characterized in that, Guanidine isothiocyanate-sulfo-SMCC (Sulfo-SMCC) crosslinking agent is used. The NHS ester at one end of the crosslinking agent reacts with the primary amine or amino-modified phalloid peptide of the secondary antibody, and the maleimide at the other end reacts with the thiol group of the coding chain after DTT reduction to form a stable covalent bond.

9. The application of a high-fidelity time-series fluorescent DNA barcode according to any one of claims 1 to 8 in the preparation of a multiplex analyzer.

10. The application of a high-fidelity time-series fluorescent DNA barcode, as described in any one of claims 1 to 8, in the preparation of a detection kit for in situ multiplex imaging and analysis of various proteins, nucleic acids, or polysaccharides.

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