Construction Of DNA Biocomputing Systems For Nucleic Acids Intelligent Sensing

With the development of science and technology,DNA is not only recognized as a carrier of genetic information but also regarded as a perfect"building material".Based on this,structural DNA nanotechnology and dynamic DNA nanotechnology have been developed.Structural DNA nanotechnology aims to construct various DNA structures through specific Watson-Crick base pairing.Such as relatively complex DNA origami composed of hundreds of strands and relatively simple DNA tetrahedron composed of a few strands.These DNA structures have advantages that single-strand DNA（ss DNA）does not have.For instance,they are stable in complex mediums and easy to enter cells.Dynamic DNA nanotechnology has developed different nucleic acid signal amplification techniques based on toehold-mediated strand displacement reaction（TMSDR）.Such as entropy-driven DNA circuits（EDC）and hybridization chain reaction（HCR）.These techniques and their cascade systems have promising applications in biosensing and bioimaging for disease markers such as micro RNA.However,in current studies,the application of various nucleic acid signal amplification technologies and their cascade systems is limited to their powerful signal amplification capabilities.The application of strict logical relations in nucleic acid intelligent sensing needs further research.DNA biocomputing system can provide an accurate output signal after simultaneously processing different input signals,making it have great application prospects in nucleic acid intelligent sensing and precise diagnosis of diseases.Therefore,utilizing the strict logical relationship of various nucleic acid signal amplification techniques to construct nucleic acid intelligent sensing systems with disease markers as input signals can expand the application range of nucleic acid signal amplification technology and provide effective means for accurate diagnosis of diseases.In addition,the following problems still exist when various DNA computing elements in DNA biocomputing system are in free state:（1）it will lead to lower DNA computing rate and efficiency.（2）Various DNA computing elements are difficult to enter cancer cells.（3）Even if various DNA computing elements are transported into cancer cells by carriers,free DNA is easily degraded by various enzymes,making it unable to perform logical computing functions.Therefore,the integration of various DNA computing elements into the DNA nanostructure that is relatively stable in complex environments and easy to enter cancer cells is particularly important for constructing efficient DNA biocomputing systems.Based on the above discussion,this thesis is committed to 1)establishing the relationship between nucleic acid signal amplification technique,DNA biocomputing,and intelligent sensing of nucleic acid,2)constructing a high-integrated DNA biocomputing system with nucleic acid disease markers as input signals to expand the application of the DNA biocomputing system in the accurate diagnosis of diseases.The main works of this thesis are as follows:1.AND logic gate-guided construction of entropy-driven DNA circuit-involved cascade system for information transferThe cascade nucleic acid signal amplification system composed of different DNA circuits has a wide range of applications in the sensitive sensing of disease markers.However,system leakage is a critical problem that limits its development.In the construction of a cascade nucleic acid signal amplification system with EDC as the upstream circuit,the EDC ternary reactant,composed of an equimolar ratio（1:1:1）of three single-strand DNA（ss DNA）in theory,was indispensable,wherein undesired system leakage occurred if there was any out-of-balance of this ratio.Herein,we proposed“splitting–reconstruction”and“protection–release”strategies on the potential downstream circuit initiator derived from upstream EDC to avoid system leakage in the construction of EDC-involved cascade circuits.Based on these two strategies,the EDC2-4WJ-TMSDR and EDC3-HCR were developed.Both the reconstructed and released downstream circuit initiator were in compliance with the principle of cascade AND logic gate.The experimental results showed that the ratio of that ss DNA is not needed to be kept at 1:1:1 to guarantee the cascade DNA system carried out in the designed order.In addition,the inherent property of upstream EDC was transmitted into the downstream circuit,endowing developed cascade circuits with more robust ability,thus achieving intelligent and sensitive sensing of DNA analog of different micro RNA.This work demonstrates great application prospects in the intelligent sensitive sensing of nucleic acid disease markers.The two strategies utilized here lay the foundation and new insight in constructing EDC-like-circuit-involved high-order nucleic acid intelligent sensors.2.Hierarchical Hybridization Chain Reaction for Amplified Signal Output and Cascade DNA Logic CircuitsIn chapter 1,the reactants of the upstream EDC were composed of three different ss DNA.The strict requirements on the proportion of the three ss DNA critically inhibited the development of the EDC-involved cascade nucleic acid signal amplification system.There was no such problem in constructing the cascade system with different circuits taking DNA hairpins as reactants.However,most studies only utilized the signal amplification function of various cascade systems.The application of the strict logical relationship between different circuits of the cascade system remains to be explored.In this work,a three-layer hierarchical hybridization chain reaction（3L h HCR）,composed of ^1stHCR,^2ndHCR,and ^3rdHCR,was proposed.A splitting-reconstruction strategy was proposed to avoid the system leakage of the 3L h HCR system.In detail,the initiator of the subordinate HCR was initially designed as two different segments（splitting）that are got together（reconstruction）for the activation of the subordinate HCR after the execution of the superior HCR.The activation of the whole 3L h HCR system brings significant fluorescence recovery by interdicting fluorescence resonance energy transfer（FRET）between fluorophore and quencher.Thus,in the range of 50 pmol/L to10 nmol/L,the ultrasensitive detection of ^1stI can be achieved.The 3L h HCR shows a better ability to distinguish against different concentrations of ^1stI than that of the 2L h HCR I system when the concentration is lower than 1 nmol/L.In addition,the hierarchical self-assembly mechanism of the 3L h HCR allows it can also be executed as a cascade AND（C-AND）logic gate with high specificity and molecular keypad lock with a prompt error-reporting function.Further,it was proved that the 3L h HCR-based molecular keyboard lock could be used for the accurate diagnosis of cancer by designing different auxiliary DNAs and using DNA analogs of three micro RNAs to open the molecular keyboard lock.This work not only provides a new idea for building a multi-level cascade nucleic acid signal amplification system and broadens the application scope of the cascade system but also shows an effective means for accurate diagnosis of diseases.3.Construction of a modular and reversible DNA computing platform for efficient nucleic acid intelligent sensingIn chapter 2,the operation of the DNA logic circuit was realized by using the cascade nucleic acid signal amplification system.However,the cross-talking between the sequence of the input signal and the signal reporting unit still limits the flexibility and scalability of the input signal sequence design in the DNA logic computing system.In addition,sensitive sensing is critical in developing logic systems with weak input signals（ISs）.In this chapter,a“weak–inputs–strong–outputs”strategy was proposed to guide the construction of sensitive logic nanodevices by integrating an input-induced reversible DNA computing platform with a HCR-based signal amplifier.By rational design computing elements（CEs）so as to avoid cross-talking between the sequence of ISs and signal amplifier,the newly constructed logic nanodevices with good sensitivity to the weak ISs even in the presence of low concentrations of CEs,and can execute YES,OR,NAND,NOR,INHIBIT,INHIBIT-OR and number classifier operation.All the experimental results demonstrated that the logic nanodevices,using signal amplifier HCR,can improve the sensitivity of the logic system to ISs.The sufficient HCR products of sensitive logic nanodevices have the potential to drive downstream logic systems to construct higher-order hierarchical circuits.In addition,the computing speed,which is significant for a robust logic system,was accelerated by employing HCR as the signal amplifier.The reversible DNA computing platform proposed in this chapter has guiding significance in the construction of efficient and universal nucleic acid intelligent sensing.4.A high-integrated DNA biocomputing platform for micro RNA intelligent sensing in living cellsIn the previous several parts,different nucleic acids intelligent sensing systems were studied at the solution level,which is still lacking in the accurate diagnosis of diseases.In this work,an aptamer-equipped high-integrated DNA biocomputing platform（HIDBP-A）that can perform AND logic operation in living cancer cells was developed by loading two different computing elements onto the same DNA tetrahedron.Two micro RNAs as input signals（micro RNA 21 and micro RNA 155 as input 1 and input 2,respectively）can activate the logical function of HIDBP-A.Aptamer AS1411 helped HIDBP-A be readily uptaken by cancer cells.When the HIDBP-A encounters the overexpressed input signals,the two computing elements suffer conformational changes and hybridize with each other through the exposure of complementary single-strand sticky ends.This generates fluorescence resonance energy transfer（FRET）between Cy3and Cy5 as a positive output signal（output 1）.Compared to the free DNA biocomputing platform（FDBP）,the integration of all computing elements into the same DNA tetrahedron greatly improves logic computing speed and efficiency.This can be explained by the confinement effect reflected by the high local concentration of computing elements.The HIDBP-A produced an output of 0 once the expression level of one or two input signals was down-regulated.As a proof of concept,the utilization of micro RNA as the input signal was beneficial for improving the scalability and flexibility of the sequence design of the logic system.Given that the different micro RNAs are overexpressed in some cancer cells,the as-proposed HIDBP-A has great value in disease accurate diagnosis.To sum up,this thesis constructs different DNA biocomputing systems for nucleic acid intelligent sensing and on this basis realizes the logical operations in cancer cells.Firstly,utilizing the cascade AND logic principle to guide the construction of a cascade nucleic acid signal amplification system.And then employing the logical relationship in the cascade nucleic acid signal amplification system to construct nucleic acids intelligent sensing system.Further through the modular design of the DNA computing platform and the introduction of signal amplifiers,an efficient and universal DNA computing system was constructed.Finally,a high-integrated DNA biocomputing platform that can be used for accurate disease diagnosis was constructed by using nucleic acids disease markers as input signals.