Design And Applications Of Intelligent DNA Logic Platforms Based On Aptamer Molecular Recognition

DNA,a new type of functional materials,is widely used in biosensing,molecular self-assembly and DNA computing.At the molecular level,DNA would be a link or bridge of molecular recognition,molecular self-assembly and molecular devices,uniting the three research frontiers.For example,DNA aptamers can specifically recognize their targets,and are widely used in the design and construction of biosensors,nucleic acid nanomachines,and molecular logic devices.Distinguishing the cell environment,analyzing,in vivo in-situ analysis and specific detection of targets,studing the physiological processes and pathological mechanisms,are very promising application prospects of these DNA devices.However,how to ensure that the probe can enter the cell and exists stably in the cell without being degraded by the intracellular nuclease?In addition,after the probe enter the cell,whether it can analyze the target with high sensitivity and selectivity?Last but not the least,whether the designed sensor only responds to the target within the specified cell instead of the target that exists outside the cells or inside other cells?How to distinguish different cellular environments?These difficulties are very critical issues that related researchs need to solve.To deal with these problems,we have developed intelligent sensors with the ability to distinguish and judge different environments.To develop such intelligent sensors,serious consideration and studies from the three angles of nucleic acid nanostructures,biosensing,and logical computing are thus keen made to solve the problems of probe cell uptake,target analysis and environmental recognition,respectively.Then,integrate them effectively into the finial design.The analyser and sensing units of DNA sensors are encapsulated in the DNA nanostructures in the design of this paper,and this design equip the analysers and sensing units with excellent cell endocytosis and biological stability.Moreover,the control units are introduced to the sensors to make it realize intelligent analysis of intracellular targets.We designed the logic circuit rationally by introducing cellular environmental factors(ATP,H+and K+etc)to make the logic circuit to be able to regulate biosensors and distinguish the internal and external environments.The research ideas and results of this paper will be significant and offer some guidance and references for personalized nanomedicine,such as intelligent diagnosis and controlled drug release.The main contents of the paper are as follows:1.Thioflavin T behaves as a fluorescent ligand for label-free ATP aptasensor.Adenosine triphosphate(ATP)mainly exists in the intracellular fluid.Although intracellular ATP level is orders of magnitude more than the extracellular environment,they keep each other in balance.Abnormal fluctuations in ATP levels can cause many human diseases.This relatively balanced concentration difference makes ATP an important physiological indicator for distinguishing the internal and external environment of cells.For most of traditional works,labeling DNA with a reporter is a prerequisite for the signal readout when constructing ATP aptasensors,which weaken the affinity of ATP aptamer(ABA27)to ATP.It also make the procedure complicated,and lead to the increase of the time-consuming,labor-intense experiment cost at the same time.The competition between ATP and thioflavin T(ThT)for ABA27 was found in our research and the mechanism was thoroughly studied.ThT,an efficient fluorescent ligand for ABA27,can bind to ABA27 and gives rise to a remarkable increase in the fluorescence intensity.ATP can replace ThT from the DNA binder ABA27,giving out a decreased fluorescence intensity.Such a displacement mechanism can be used to constructed a label-free ATP aptasensor,with ThT as a specific fluorescent ligand for signal readout.By this means,sensitive ATP detection was achieved with a high selectivity.The detection limit of is 5 ?M,which is comparable with maximum concentration of extracellular ATP.The sensing strategy provides a novel signal readout mode for label-free selective ATP detection.2.The design of environment-responsive intelligent DNA logic circuit platforms for controllable molecular sensing.Focusing on the problems of environmental recognition and judgment faced by DNA aptamer sensors for in-situ analysis applications,here we report a simulated cellular environment-responsive DNA logic circuit for controllable molecular sensing.Two intrinsic cellular components(ATP and K+)were used as the inputs due to their remarkable concentration differences outside and inside cells.We designed an H-shape scaffold.By assembling ATP aptamer and G-quadruplex into the H-shaped DNA scaffold,intelligent DNA logic circuits,that can recognize cell-simulated bioenvironments and modulate the operations of a DNA nanosensor,were sucessfully constructed.We integrate the analysis elements of ATP.K+and DNA into H-shaped DNA nanostructures.Based on toehold-mediated strand displacement(TMSD)and target-induced DNA allosteric,the H-shaped DNA nanostructures can respond to ATP.K+and DNA.controllably.Without changing any input substances.we have successfully constructed "AND-AND","OR-AND","AND-OR" and "OR-OR" four different logic circuits by changing the H-shaped DNA scaffold slightly.In addition,we applied the H-shape nanostructure to recognize simulated cell environments and to analyze the nucleic acids in MCF-7 cell lysate buffer.The H1 structure receive some information relating to ATP and K+,and determine whether it is an intracellular environment or an extracellular environment after processing the information.Then,it can feedback information to the secondary nucleic acid sensor to control the interaction between the nucleic acid nanostructure and the simulated intracellular nucleic acid.The H-shaped DNA nanostructures and the methods for constructing multiple logic circuits provide new ideas for the design of smart logic devices.With the ability to recognize simulated cell environments,the intelligent DNA logic circuit nucleic acid analysis device is of great significance to biomedical researches,such as targeted drug delivery and programmable molecular sensing.3.Cellular environment-recognizing DNA-computation circuits for the intracellular transport of molecular payloads for mRNA imaging.Intracellular delivery,in situ activation and controlled release of molecular loads,including proteins,anticancer drugs,siRNAs,and ASOs,remain a huge challenge in biomedicine.We suppose using TK1 mRNA as target,FNA nanostructure as nanocarrier,and employ endogenous cellular components(ATP and H+)instead of external stimuli to activate FNA nanocarriers in situ and unload molecular payloads logically controlled by cellular environment-responsive DNA-computation circuits for mRNA imaging.To bring this idea to reality,we integrated functional DNA based logic circuits with FNA nanocarriers that consist of a truncated square pyramid(TSP)cage with an ATP aptamer(ABA27)and i-motif in its two edges,and a built-in ASO containing duplex as the sensing element of TK1mRNA.Two cascaded logic gates(OR-AND and AND-AND)are constructed on the FNA scaffold to variably respond to ATP and protons.After FNA endocytosis into the target cell,the endogenous ATP molecules and H+can effectively control the ASO double-strand to release from the TSP nanocage and downstream interaction with TK1 mRNA,generating a fluorescent signal for intracellular TK1 mRNA imaging.This approach would be further applied to regulating mRNA-based therapeutics in tumor tissues with abnormal pH and ATP levels.