Research On Molecular Circuits Based On Aptamer And Transcription

Due to their excellent operability and sequence programmability,molecular circuits have been applied in many fields such as DNA computing,gene detection,disease diagnosis,and intelligent nano-systems.Integrating RNA molecules,metal ions,enzymes,or even entire cells into circuits to achieve biocompatible molecular systems is an urgent problem to be solved.The transcription performs the information transfer from DNA to RNA.Similar to the naturally occurring gene regulatory network,it is a promising tool to construct complex dynamic circuits from the bottom up.Aptamers are artificially screened DNA or RNA molecules,which can bind to various non-nucleic acid target molecules with high specificity and affinity.The aptamer provides an efficient way to integrate non-nucleic acid molecules into DNA circuits.This paper concentrates on developing easily integrated and programmable circuit elements based on aptamers and in vitro transcription to expand the application scope of molecular circuits.The main work is as follows.To address the lack of diversity in the regulation of in vitro transcription circuits,a regulatory strategy based on aptamer was proposed.The regulation was implemented by controlling the conformation of the aptamer structure via multiple biomolecules,including DNA,RNA,restriction enzymes,and methylase.Based on this strategy,the activation and inhibition of the transcription circuits were performed.A two-level cascading network and an enzyme-controlled switch circuit were also established.The gel electrophoresis and fluorescence experiments demonstrated that the proposed strategy was flexible,scalable,and expanded the in vitro synthetic biology toolkit.To overcome the limitation of programmability in the double-helix aptamer structure,a strategy based on a clamp triple-stranded aptamer structure(CLTAS)was proposed.The method switched DNA polymerase activity through conformational changes of the CLTAS and then further activated or inhibited the transcription circuit.The programmable regulation was performed by changing structural parameters such as arm length,GC content,and overhang position of the CLTAS.The dynamic reactions,such as CLTAS-based strand displacement or enzyme cleavage,were also used to regulate transcription circuits.Based on CLTAS,a biosensing circuit and hybrid logic circuits were constructed.The experimental results demonstrated that the proposed CLTAS strategy was programmable,controllable,and provided high affinity and specificity for identifying target molecules,having potential in the fields of biosensing,biocomputing,and programmable nanomachines.To solve the problem of lacking the programmable multi-responsive components in the cell-free in vitro transcription circuit,an approach was proposed to construct molecular circuits by regulating the promoter region of the transcription template.The transcription circuits could process variable inputs by optimizing the transcription template's linear structure and multi-way junction structure.Logic circuits and cascading circuits were also realized.The experimental results demonstrated that the proposed method had the advantages of modularity,scalability,and provided programmable and composable modules for bioanalytical and biocomputing applications.To solve the single function and uncontrollability of DNA sensors,a method for engineering structure-switching biosensors was proposed.The engineered biosensor could respond to external stimuli while preserving the sensing capability by adding an extra actuation module.As a demonstration of this method,a controllable Hg²⁺sensor was designed,in which logic gate was employed as the actuation module.Thus,multiple recognition of Hg²⁺and oligonucleotides was achieved.Experimental results demonstrated that the proposed method had the advantages of versatility and modularity,making it possible to control other structure switching sensors such as ATP sensors.To solve the problem of single regulatory factors in the strand displacement-based molecular circuit,a cofactor-assisted three-way junction-driven strand displacement strategy was proposed,which could tune the reaction kinetics by the collaboration of DNA and other types of stimulus.This strategy is responsive to various inputs by inserting the aptamer sequence into the three-way junction structure.To demonstrate the strategy,adenosine triphosphate(ATP),HG²⁺,and p H were used as cofactors to modulate the displacement reaction,and the corresponding multi-input AND logic circuit was established.Experimental results demonstrated that the proposed method provided design flexibility for constructing multi-input dynamic nanodevices and dynamic DNA nano-systems.