Accurate construction strategy of high-sensitivity DNA circulating induction type fluorescent nano-robot

Abstract

The invention discloses a precise construction strategy of a high-sensitivity DNA circulation inducible fluorescent nanorobot, which specifically comprises the following steps: A1, constructing a DNA nanostructure framework which is composed of a modularly designed recognition domain, a signal conversion domain and a circulation trigger domain, the recognition domain comprises an aptamer or a hairpin structure and is used for specifically combining with a target molecule, and the circulation trigger domain is used for specifically combining with the target molecule; the invention relates to the technical field of gene engineering. According to the precise construction strategy for the high-sensitivity DNA circulating inducible fluorescent nano-robot, through integration of a modular DNA tetrahedral framework and an enzyme digestion-strand displacement synergistic amplification system, cyclic utilization and signal exponential gain (such as CHA cascade amplification combined with CRISPR-Cas12a trans-cleavage) of a target object are achieved, the detection limit breaks through the 0.1 am level, and the accuracy of the high-sensitivity DNA circulating inducible fluorescent nano-robot is greatly improved, so that the accuracy of the high-sensitivity DNA circulating inducible fluorescent nano-robot is improved, and the accuracy of the high-sensitivity DNA circulating inducible fluorescent nano-robot is improved. Compared with a traditional single enzyme digestion or linear amplification method, the sensitivity is improved by at least three orders of magnitude. Meanwhile, time-resolved upconversion nanoparticles (UCNPs) are combined with a magnetic bead separation-lock type probe noise reduction technology.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and specifically to a precise construction strategy for highly sensitive DNA cycle-inducible fluorescent nanorobots. Background Art

[0002] In the 1950s, JD Watson and FHCC Rick published the double helix structure model of DNA in Nature, suggesting that DNA is the genetic template for the inheritance of life. In 1983, Seeman first used DNA to construct nucleic acid nanostructures, demonstrating that DNA not only carries important genetic information for life, but can also be used as a component for the construction of nanomaterials.

[0003] According to the patent document, titled "Construction Method of DNA Cycle-Induced Opening DNA Fluorescent Nanorobot" (Patent Publication Number: CN109338014A, Patent Publication Date: 2019-02-15), for the first time, a DNA nanorobot that drives DNA cycle-induced opening is combined with metal nanoclusters synthesized in situ using DNA as a template to construct a new type of fluorescent nanorobot, achieving label-free, modification-free, and fluorescence signal induction cascade enhancement functions. This provides new ideas for the rational design of functional DNA nanorobots and a new tool for fluorescence imaging technology. The use of this multifunctional nanorobot for target detection has not been reported before, expanding the application of functional DNA nanorobots and providing new ideas for biosensor technology. The construction of a new label-free, DNA cycle-induced opening fluorescent nanorobot improves the working efficiency of the nanorobot and achieves fluorescence label-free.

[0004] Based on the description in the above documents, existing traditional DNA nanorobots are susceptible to background noise interference in complex biological samples, and rely on single enzyme cleavage or static signal amplification strategies, with limited sensitivity, making it difficult to meet the detection needs of ultra-low abundance markers. To this end, the present invention provides a precise construction strategy for highly sensitive DNA circulation-induced fluorescent nanorobots. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a precise construction strategy for highly sensitive DNA loop-induced fluorescent nanorobots, which solves the problem that traditional DNA nanorobots are easily interfered by background noise in complex biological samples, rely on single enzyme cleavage or static signal amplification strategies, have limited sensitivity, and are difficult to meet the detection needs of ultra-low abundance markers.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: a precise construction strategy for a highly sensitive DNA recycling-inducible fluorescent nanorobot, specifically comprising the following steps:

[0007] A1. Constructing a DNA nanostructure framework consisting of a modularly designed recognition domain, a signal transduction domain, and a loop triggering domain. The recognition domain contains an aptamer or hairpin structure for specific binding to a target molecule.

[0008] A2. Construct dynamic regulatory elements to remotely control the conformational switching of nanorobots through light-responsive hinges or pH-responsive structures in the dynamic regulatory elements.

[0009] A3. Construct a closed-loop circulation system to achieve the recycling of the target product through the entropy-driven chain displacement network or enzyme cleavage-chain displacement synergy of the closed-loop circulation system.

[0010] A4. Construct a fluorescence reporter system and achieve signal output and background noise suppression by integrating time-resolved upconversion nanoparticles or multicolor quantum dots into the fluorescence reporter system.

[0011] Preferably, the DNA nanostructure framework is a tetrahedral framework formed by annealing four single-stranded DNAs, and the recognition domain, signal conversion domain and loop trigger domain are anchored at the three vertices of the tetrahedron respectively, with a spatial spacing of 5-10 nanometers.

[0012] Preferably, the dynamic regulatory element is an azobenzene-modified double-stranded DNA hinge, which can be reversibly unwound by 365 nm ultraviolet light irradiation, triggering the opening and closing of the hairpin structure of the signal transduction domain.

[0013] Preferably, the closed-loop circulation system comprises a nickase recognition site and a catalytic hairpin assembly primer sequence. The target triggers enzyme cleavage to release a short-chain primer, driving CHA cascade amplification to generate a double-stranded DNA product, thereby achieving signal exponential amplification.

[0014] Preferably, in the fluorescence reporter system, the time-resolved upconversion nanoparticles (UCNPs) are NaYF4:Yb 3+ / Tm 3+ , coupled with the Cy3 fluorescent group through FRET, and the distance between the two is less than 10nm; a time-resolved fluorescence instrument is used for detection, and the delay time is 50-200 microseconds.

[0015] Preferably, the fluorescent reporter system further comprises a magnetic bead separation module, wherein the surface of the magnetic beads is coupled with biotinylated capture probes for pre-enrichment of the target, and non-specific signals are suppressed by padlock probes and rolling circle amplification (RCA).

[0016] Preferably, the fluorescent reporter system comprises a multi-color quantum dot encoding module, and CdSe / ZnS quantum dots are connected to DNA walker probes through a thiol-maleimide reaction to achieve multi-target synchronous detection.

[0017] Preferably, the nanorobot is encapsulated in a droplet with a diameter of 10 microns, and the single droplet signal is counted in real time by a high-speed fluorescence imager, with a detection limit of 0.1am and a signal-to-noise ratio of ≥50:1.

[0018] Beneficial effects

[0019] The present invention provides a precise construction strategy for highly sensitive DNA cycle-induced fluorescent nanorobots.

[0020] Compared with the existing technology, it has the following beneficial effects:

[0021] 1. This precise construction strategy for highly sensitive DNA recycling-induced fluorescent nanorobots achieves target recycling and exponential signal gain (such as CHA cascade amplification combined with CRISPR-Cas12a trans-cleavage) through the integration of a modular DNA tetrahedron framework and an enzyme cleavage-strand displacement synergistic amplification system. The detection limit has been broken through to 0.1am, which is at least 3 orders of magnitude higher than the sensitivity of traditional single enzyme cleavage or linear amplification methods. At the same time, the combination of time-resolved upconversion nanoparticles (UCNPs) and magnetic bead separation-lock probe noise reduction technology effectively eliminates short-lived autofluorescence and nonspecific amplification interference in complex samples such as serum, and the signal-to-noise ratio is improved to ≥50:1.

[0022] 2. This precise construction strategy for a highly sensitive DNA-loop-inducible fluorescent nanorobot incorporates a photoresponsive azobenzene hinge and an entropy-driven chain displacement network. This allows for remote control of the nanorobot's conformational switching via ultraviolet light, enabling on-demand activation and deactivation of signaling pathways and preventing the accumulation of false positives from non-target triggering. The closed-loop system, through target regeneration and a self-sustaining fuel chain, ensures sustained signal output for >6 hours, achieving over 10 times greater stability than conventional single-shot triggering systems.

[0023] 3. This precise construction strategy for highly sensitive DNA-loop-inducible fluorescent nanorobots utilizes a multicolor quantum dot encoding module and microfluidic droplet single-molecule encapsulation technology to simultaneously differentiate and detect multiple targets (such as miRNA-21 and let-7a) in a single system. Single-droplet fluorescence imaging statistically eliminates population averaging errors, achieving single-molecule resolution. Furthermore, optimized FRET design and precise control of DNA tetrahedron spacing ensure fluorescence energy transfer efficiencies exceeding 80%, minimizing spectral crosstalk. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a construction flow chart of the present invention. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] See also Figure 1 , the present invention provides two technical solutions:

[0027] The precise construction strategy of highly sensitive DNA loop-inducible fluorescent nanorobots includes the following steps:

[0028] A1. Constructing a DNA nanostructure framework consisting of a modularly designed recognition domain, a signal transduction domain, and a loop triggering domain. The recognition domain contains an aptamer or hairpin structure for specific binding to a target molecule.

[0029] A2. Construct dynamic regulatory elements to remotely control the conformational switching of nanorobots through light-responsive hinges or pH-responsive structures in the dynamic regulatory elements.

[0030] A3. Construct a closed-loop circulation system to achieve the recycling of the target product through the entropy-driven chain displacement network or enzyme cleavage-chain displacement synergy of the closed-loop circulation system.

[0031] A4. Construct a fluorescence reporter system and achieve signal output and background noise suppression by integrating time-resolved upconversion nanoparticles or multicolor quantum dots into the fluorescence reporter system.

[0032] In this embodiment, the DNA nanostructure framework is a tetrahedral framework formed by annealing four single-stranded DNAs. The recognition domain, signal conversion domain, and loop trigger domain are anchored at the three vertices of the tetrahedron, respectively, with a spatial spacing of 5-10 nanometers.

[0033] The modularly designed DNA tetrahedron framework (with the recognition domain, signal conversion domain, and loop trigger domain precisely anchored at three vertices with a spacing of 5-10 nm) achieves efficient synergy of functional modules and optimized signal transmission. The rigid tetrahedral structure avoids the random folding problem of traditional linear DNA probes, ensuring an initial quenching efficiency of >95% for the fluorophore (Cy3) and quencher (BHQ2), significantly reducing background noise.

[0034] In this embodiment, the dynamic regulatory element is an azobenzene-modified double-stranded DNA hinge, which can be reversibly unwound by 365-nanometer ultraviolet light irradiation, triggering the opening and closing of the hairpin structure of the signal transduction domain.

[0035] The introduction of an azobenzene-modified double-stranded DNA hinge allows for reversible unwinding upon 365nm UV light, enabling remote and precise control of the nanorobot's conformation. This light-controlled switch can shorten the on / off time of the signal pathway to seconds (compared to minutes for conventional pH responses), and improves cyclic stability to >100 switching cycles without attenuation.

[0036] In this embodiment, the closed-loop circulation system includes a nickase recognition site and a catalytic hairpin assembly primer sequence. The target triggers enzyme cleavage to release a short-chain primer, driving CHA cascade amplification to generate a double-stranded DNA product, thereby achieving signal exponential amplification.

[0037] Through the synergistic effect of the nickase (Nt.BbvCI) recognition site and catalytic hairpin assembly (CHA), a single target trigger can generate 10 3 The PCR-based enzyme digestion and strand displacement cascade reaction reduces the detection limit to 0.1 μm, a 100-fold increase in sensitivity compared to traditional single enzyme digestion strategies.

[0038] In this embodiment, the time-resolved upconversion nanoparticles (UCNPs) in the fluorescence reporter system are NaYF4:Yb 3 + / Tm 3+ , coupled with the Cy3 fluorescent group through FRET, and the distance between the two is less than 10nm; a time-resolved fluorescence instrument is used for detection, and the delay time is 50-200 microseconds.

[0039] Integrated NaYF4:Yb 3+ / Tm 3+ The FRET system of upconversion nanoparticles (UCNPs) and Cy3 uses time-resolved fluorescence detection (delay of 50-200μs) to filter out short-lived background interference (such as serum autofluorescence), improving the signal-to-noise ratio to ≥50:1. The 980nm excitation light from UCNPs avoids photodamage to biological samples, making it suitable for real-time monitoring in vivo.

[0040] In this embodiment, the fluorescent reporter system further includes a magnetic bead separation module. The surface of the magnetic beads is coupled with biotinylated capture probes for pre-enrichment of the target, and nonspecific signals are suppressed by padlock probes and rolling circle amplification (RCA).

[0041] Streptavidin magnetic beads pre-enrich the target, and combined with padlock probes and rolling circle amplification (RCA) technology, the nonspecific signal suppression rate exceeds 90%, and the target capture efficiency reaches 95%. Magnetic bead separation increases the detection sensitivity of rare samples (such as circulating tumor DNA) by 10 times.

[0042] In this embodiment, the fluorescent reporter system includes a multi-color quantum dot encoding module, and CdSe / ZnS quantum dots are connected to DNA walker probes through a thiol-maleimide reaction to achieve multi-target simultaneous detection.

[0043] CdSe / ZnS quantum dots (QD605, QD705) are coupled to DNA walker probes through a thiol-maleimide reaction to achieve simultaneous detection of multiple targets (such as miRNA-21 and let-7a) in a single tube. The error in channel signal acquisition is less than 1%, and the detection efficiency is increased by 5 times.

[0044] In this embodiment, the nanorobot is encapsulated in a droplet with a diameter of 10 microns, and the single droplet signal is counted in real time by a high-speed fluorescence imager, with a detection limit of 0.1am and a signal-to-noise ratio of ≥50:1.

[0045] The nanorobots were encapsulated in 10μm droplets (single droplet volume 0.5pL), and single-molecule events were counted using a high-speed fluorescence imager, achieving a detection limit of 0.1 μm (compared to 1 fM with traditional methods) and reducing sample consumption to 1 / 1000. Droplet isolation effectively prevents cross-contamination and is suitable for single-cell analysis.

[0046] In summary, the target (such as miRNA-21) binds to the recognition domain aptamer, triggering the opening of the hairpin structure, and Cy3 moves away from the BHQ2 quencher, releasing the initial fluorescence signal. Ultraviolet light (365nm) irradiates the azobenzene hinge, unwinding the double-stranded DNA, forcing the hairpin structure to close, achieving remote control of the signaling pathway. After the hairpin opens, the Nt.BbvCI enzyme cleavage site is exposed, and the enzyme cleaves and releases the short chain primer. The primer triggers the CHA reaction to generate a large amount of double-stranded DNA, and at the same time releases the target to enter the next round of cycles. Each cycle produces 10 3 The signal gain is 10-fold, and combined with CRISPR-Cas12a trans-cleavage, the signal is further amplified to 10 6 times. UCNPs emit 365nm light under 980nm excitation, transferring energy to Cy3 (570nm fluorescence) through FRET. The time-resolved mode (delay 50-200μs) filters out short-lived background fluorescence. Magnetic bead pre-enrichment combined with lock probe-RCA technology eliminates nonspecific adsorption in serum; quantum dot channel detection (605nm / 705nm) avoids spectral overlap. Microfluidic droplet encapsulation achieves single nanorobot isolation, and by counting the fluorescence positivity rate of thousands of droplets (0.1am corresponds to approximately 1 positive droplet / μL), the single-molecule detection limit is achieved.

[0047] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0048] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0049] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A precise construction strategy for highly sensitive DNA loop-inducible fluorescent nanorobots, characterized by: The specific steps include: A1. Constructing a DNA nanostructure framework consisting of a modularly designed recognition domain, a signal transduction domain, and a loop triggering domain. The recognition domain contains an aptamer or hairpin structure for specific binding to a target molecule. A2. Construct dynamic regulatory elements to remotely control the conformational switching of nanorobots through light-responsive hinges or pH-responsive structures in the dynamic regulatory elements. A3. Construct a closed-loop circulation system to achieve the recycling of the target product through the entropy-driven chain displacement network or enzyme cleavage-chain displacement synergy of the closed-loop circulation system. A4. Construct a fluorescence reporter system and achieve signal output and background noise suppression by integrating time-resolved upconversion nanoparticles or multicolor quantum dots into the fluorescence reporter system.

2. The precise construction strategy of the highly sensitive DNA recycling-inducible fluorescent nanorobot according to claim 1 is characterized by: The DNA nanostructure framework is a tetrahedral framework formed by annealing four single-stranded DNAs. The recognition domain, signal conversion domain and loop trigger domain are anchored at the three vertices of the tetrahedron respectively, and the spatial spacing is 5-10 nanometers.

3. The precise construction strategy of the highly sensitive DNA recycling-inducible fluorescent nanorobot according to claim 1 is characterized by: The dynamic regulatory element is an azobenzene-modified double-stranded DNA hinge, which can be reversibly unwound by 365-nanometer ultraviolet light irradiation, triggering the opening and closing of the hairpin structure of the signal conversion domain.

4. The precise construction strategy of the highly sensitive DNA recycling-inducible fluorescent nanorobot according to claim 1 is characterized by: The closed-loop circulation system includes a nickase recognition site and a catalytic hairpin assembly primer sequence. The target triggers enzyme cleavage to release a short-chain primer, driving CHA cascade amplification to generate double-stranded DNA products, achieving signal exponential amplification.

5. The precise construction strategy of the highly sensitive DNA recycling-inducible fluorescent nanorobot according to claim 1 is characterized by: In the fluorescence reporter system, the time-resolved upconversion nanoparticles (UCNPs) are NaYF4:Yb 3+ / Tm 3+ , coupled with the Cy3 fluorescent group through FRET, and the distance between the two is less than 10nm; a time-resolved fluorescence instrument is used for detection, and the delay time is 50-200 microseconds.

6. The precise construction strategy of the highly sensitive DNA recycling-inducible fluorescent nanorobot according to claim 1 is characterized by: The fluorescent reporter system also includes a magnetic bead separation module. The surface of the magnetic beads is coupled with biotinylated capture probes for pre-enrichment of the target, and non-specific signals are suppressed by padlock probes and rolling circle amplification (RCA).

7. The precise construction strategy of the highly sensitive DNA recycling-inducible fluorescent nanorobot according to claim 1 is characterized by: The fluorescent reporter system includes a multi-color quantum dot encoding module, and CdSe / ZnS quantum dots are connected to DNA walker probes through a thiol-maleimide reaction to achieve multi-target synchronous detection.

8. The precise construction strategy of the highly sensitive DNA recycling-inducible fluorescent nanorobot according to claim 1 is characterized by: The nanorobot is encapsulated in a 10-micron diameter droplet, and the single droplet signal is counted in real time by a high-speed fluorescence imager, with a detection limit of 0.1am and a signal-to-noise ratio of ≥50:1.

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