Construction Of DNA Logic Gate Devices And Their Application In Tumor Cell Imaging

The development of novel strategies and methods for tumor cell imaging with high sensitivity,high specificity,high efficiency and high biosafety is of great significance for accurate diagnosis and treatment of tumors.In recent years,DNA logic gate devices,as a kind of nanodevices based on DNA materials that can perform Boolean logic operations,have attracted much attention in the field of tumor cell imaging.On the one hand,based on Boolean logic operation,DNA logic gate devices can detect and analyze multiple tumor biomarkers simultaneously,providing more intelligent,convenient and accurate tumor cell identification and imaging services.On the other hand,with DNA as the basic construction material,DNA logic gate devices have the advantages of strong designability,simple preparation and good biological safety,which can meet the needs of tumor cell imaging under different situations and environments.At present,a variety of tumor biomarker-driven DNA logic gate devices have been designed and developed.In particular,DNA logic gate devices designed based on cell membrane surface work have shown incomparable advantages in tumor cell imaging.However,these DNA logic gate devices still faces some challenges in practical applications,mainly as follows:(1)the logical response unit is often fails to perceive the target in time due to insufficient sensitivity,resulting in false negative results;(2)The accuracy of tumor cell imaging is limited by the same type of tumor biomarkers as logical driving targets;(3)In the process of tumor cell identification,it is impossible to overcome the problem of low sensitivity caused by the weakening or loss of signal during logic operation;(4)Low speed and efficiency of logical calculation,unable to achieve fast and efficient tumor cells imaging.Based on this,this paper addresses the abovementioned challenges faced by DNA logic gate devices in tumor cell imaging by rationalizing the use of tumor cell membrane proteins and extracellular environmental factors as logic-driven targets,combined with the continuous development of functional nucleic acid(Aptamer and i-motif)with special biological significance and nucleic acid signal amplification techniques and spatial domain-limiting effects,etc.A series of novel DNA logic gate devices with high sensitivity,high specificity,high efficiency and high biological safety were constructed for the analysis and imaging of tumor cells.The detailed description is listed as follows:1.Acid-stimulus-responsive “mimosa” DNA logic gate device for sensitive sensing of extracellular pH of tumor cellsIn order to obtain DNA logic gate devices that can respond sensitively to pH dynamic changes in the microenvironment of tumor cells,the pH response interval was regulated by a partially complementary DNA strand(B-strand)through reasonable modification of pH-responsive i-motif.It has the ability to respond to small pH changes in the microenvironment of tumor cells.Then,AS1411 Aptamer was used as the membrane protein recognition anchor unit.Through reasonable design and assembly,a “mimosa” DNA logic gate device(DNM)anchored to cell membrane surface was obtained.The AS1411 Aptamer domain of DNM can bind to specific membrane proteins on the surface of target tumor cells.At the same time,due to low pH stimulation,the i-motif fragment forms a tetramer,releasing B-strand and causing the DNM to become closed and output FRET signals,which is similar to the behavior of mimosa in response to external physical stimuli.The results showed that the DNM exhibits excellent specificity and sensitive pH response(minimum 0.50 pH)inspired by Boolean operations of logic gates and “stimulus response”behavior of mimosa,providing an ideal tool for tumor cell imaging and extracellular pH sensing.2.Multi-target driven “AND” DNA logic gate device for extracellular pH/ATP imaging of tumorsIn the previous work,we constructed a DNA logic gate device that could sensitively respond to dynamic pH changes in the extracellular environment of tumors through clever design,and verified the feasibility of tumor cell membrane proteins and extracellular pH driven DNA logic gate devices for tumor cell analysis and imaging.Basis on this,this work further explores the use of multi-type and multi-dimensional tumor biomarkers to drive DNA logic gate devices to improve the accuracy of tumor cell imaging.By using ATP Aptamer as specific ATP response unit,i-motif as pH response unit,and AS1411 Aptamer as membrane protein recognition anchor unit,an “AND”DNA logic gate device(YAP)with pH,ATP,and membrane protein co-driven was constructed.The YAP device is used for precision analysis and imaging of tumor cells.Specifically,the YAP is anchored to the membrane surface of target tumor cells with the help of the AS1411 Aptamer structural domain.Then,stimulated by extracellular acidic pH and ATP,the i-motif and ATP Aptamer domains in YAP undergo configuration changes and are released from YAP respectively,which separates the quencher BHQ2 in YAP from the fluorophore Cy5,and Cy5 emits strong fluorescence signals.Thus,the “AND”logic analysis and fluorescence imaging of target tumor extracellular pH and ATP can be realized.The results showed that YAP exhibited sensitive response in the range of pH 6.2-6.8 and 100-500 μM ATP concentration,and could perform the “AND” logic operation on the surface of MCF-7 cell membrane(target tumor cell),and the logic analysis of extracellular pH and ATP.In addition,YAP was able to take full advantage of the multi-targetdriven “AND” logic gate to achieve accurate fluorescence imaging of specific target tumor cells in the eight model tumor cells constructed.With novel design and powerful functions,the DNA logic gate device is expected to provide an effective tool for basic medical research and accurate diagnosis of diseases.3.Membrane protein and extracellular pH co-driven amplified DNA logic gate device for precise identification and imaging of tumor cellsIn the previous work,we constructed a multi-target-driven “AND” DNA logic gate device,which realized the logical analysis of tumor extracellular factors(pH and ATP)and accurate imaging of tumor cells.However,in practical applications,a challenge remains that DNA logic gate devices respond to targets on complex cell membranes and perform logic operations in which there is unavoidable weakening and loss of logic operation signals in the process,resulting in low sensitivity of tumor cell detection and imaging.In order to solve this problem,a DNA logic gate device based on HCR signal amplification technology was designed in this work(HDLG).Specifically,imotif and Aptamer are combined as the logic trigger switch unit,and the HDLG can be driven to perform the “AND” logic operation only in the presence of acidic pH and membrane protein,and then induce the HCR to output an amplified “ON” fluorescent signal on the cell surface.The results showed that the HDLG can achieve autonomous logical computation and highresolution fluorescence imaging on the surface of model target cell(CEM).Thanks to the HCR signal amplification design,the HDLG has excellent sensitivity and specificity,not only able to detect a minimum of 70 target cells in 200 μL PBS,but also enable accurate fluorescence labeling of target cells in complex cell mixtures.Compared with the traditional DNA nanodevices,the novel HCR signaling large DNA logic gate devices will have a wider application prospect in the fields of biomedicine and personalized medicine.4.Multi-target-driven confined effector DNA logic gate device for accurate recognition and efficient imaging of tumor cellsIn the previous work,we combined HCR signal amplification technology and used i-motif and Aptamer as the logic trigger switch unit to construct a tumor extracellular pH and membrane protein co-driven signal amplification large DNA logic gate device,which realized highly sensitive detection and amplification imaging of tumor cells.However,since the response process of this DNA logic gate device is engaged by the HCR hairpin probe as well as multiple diffusion-prone logic response elements,which leads to problems such as low speed and efficiency of logic operation in solution phase,and limits its wider application.In order to further improve the efficiency and accuracy of tumor cell imaging,functional nucleic acids(Aptamer and i-motif)responding to membrane proteins,extracellular ATP and pH were simultaneously integrated on a long Y-shaped scaffold with DNA hairpins amplifying HCR signals to generate spatially confinement effect.A confined effector multi-target driven DNA logic gate device(DMP)was developed for accurate recognition and efficient imaging of tumor cells.In this work,DMP can be activated only when the target tumor cells meet the requirements of membrane proteins,extracellular acidic pH and ATP at the same time,and then trigger the HCR confined to the long Y-shaped scaffold,benefiting from the spatial confinement effect to efficiently achieve amplified fluorescence imaging on the surface of target tumor cell membranes.The results showed that DMP can perform fast autonomous logic operation on the surface of model target cell(CEM),and achieve amplified fluorescence imaging in only60 minutes.In addition,DMP can not only accurately identify target tumor cells in the eight model tumor cells constructed,but also fluorescently label target cells in complex cell mixtures with precision and efficiency.As an intelligent,accurate and efficient DNA logic gate device,DMP is expected to provide a new perspective for the development and development of tumor diagnosis and treatment platform.