Construction And Applications Of Functional DNA Molecular Devices

As a new class of functional material,DNA molecular devices have developed greatly over the past ten years.They have a variety of types with a wide range of applications.DNA molecular devices not only play significant roles for life science research,but also show great importance in the fields of energy,information,computer chips,etc.Compared with traditional materials,functional DNA molecular devices have advantages such as better stability,programmability,and biocompatibility,etc.In addition,they have been widely utilized for biosensing,cell or tissue imaging,drug delivery,and disease precision treatment and other fields.Based on the structural features and basic working mechanisms of functional DNA molecular devices,we have designed and constructed several functional DNA molecular devices here and utilized them for ion or molecular sensing,subcellular fluorescence imaging,and logic transmembrane transport of target molecules,etc.This paper includes the following parts:(1)Construction and sensing applications of DNA molecular devices based on the fluorescence enhancement effect of looped-out 2-aminopurine.Since 2-aminopurine is an analogue of adenine,it is able to replace adenine to hybridize with its complementary base.We have made a systematically research and found that the fluorescence of 2-aminopurine was closely related to its base environment,including the loop size and the neighboring bases.Generally,it shows a higher fluorescence emission intensity with less neighboring bases.For example,2-aminopurine has much higher fluorescence when located in the shorter loops in the DNA double strand and G-quadruplexes.In addition,the fluorescence of 2-aminopurine is much higher in the propeller-like loops than in diagonal and lateral ones of G-quadruplexes.Based on the fluorescence enhancement effect of the looped-out 2-aminopurine,we designed a series of DNA molecular devices,including an HBV gene sensor by hybridization chain reaction(HCR),a DNA triplex switch that responds to pH dynamically,and a reversible unlabeled molecular sensor for detection of K+and Pb2+.These sensors exhibit high specificity and sensitivity towards these targets and have broad potential for future construction of DNA molecular devices and sensing applications.(2)A DNA nanoswitch-controlled reversible nanosensor.DNA molecular switches can be regulated by external stimuli to make continuously conformational changes between two or more states reversibly.These external stimuli include electrons,protons,photons,pressure,magnetism and temperature,etc.Based on these properties of DNA molecular switches,we proposed a novel reversible molecular sensor controlled by a DNA molecular switch,whose scaffold is formed by the hybridization of three single-stranded DNAs containing ATP aptamers.And the processes of capturing and releasing of ATP molecules are conducted through toehold-mediated strand displacement reaction.In addition,Thioflavin T(ThT)is utilized as an indicator for ATP binding and release,and reflects the process of ATP binding and release through the decrease of the fluorescent signal.The proposed sensor not only responds to ATP molecules with a detection limit of 10 μM,but also switches reversibly between the "OFF" and "ON" states through DNA displacement reactions.We envision this sensor has broad potential for designing reversible nanomachines,nanocarriers for drug delivery and other related DNA nanodevices.(3)Reconfigurable bioinspired framework nucleic acid nanoplatform dynamically manipulated in living cells for subcellular imaging.The pH-sensitive N-terminal of spider silk proteins(spidroins)plays a vital role for the dimerization of spidroins:they are dimerized to form the head-to-tail antiparallel dimers upon lowering the pH.Such a pH-dependent dimerization behavior is also observed in DNA self-assembly guided by the i-motif.Given the common pH-dependent control,here we design two different i-motif-programmed framework nucleic acid(FNA)units that are dynamically heterodimerized in acidic lysosomes within living cells to mimic the initial step of spidroin assembly.In addition,we further tethered split ATP aptamers on FNAs to bind ATP molecules.In this way,we can use laser confocal microscopy to achieve ATP imaging at the subcellular level.Given the similar pH-responsive behavior,we envisage engineering an artificial "hybrid" silk fiber with tailored properties by replacing the N-terminal,which has broad potential for biomimics.(4)Extracellular ion-responsive logic sensors utilizing DNA dimeric nanoassemblies on cell surface and application to boosting AS1411 internalization.The tumor microenvironment is known to be featured by high level of extracellular potassium ions and increased acidity.Based on these properties of extracellular environment,we have built a framework nucleic acids(FNA)logic nanoplatform to logically responding to extracellular H+ and K+and improve the tumor-targeting effect drugs for improving the precision of treatment.We integrate a functional DNA-based logic circuit with an FNA nanoplatform that consists of two tetrahedral DNA nanostructures(TDNs)with different branched vertexes where the anticancer AS 1411 aptamer and a bimolecular i-motif are tethered.When attached to cancer cell surface by cholesterol anchorage,the FNA platform logically responds to the extracellular surroundings,resulting in a dimeric assembly and meanwhile AS1411 release.In addition,It enables a proximity DNA hybridization-based FRET to localize plasma membrane boundary and thus benefit to fluorescently tracking internalized AS 1411,which collectively behaves like a logic circuit consisting of tandem YES and AND gates.In contrast to a low delivery efficiency of AS 1411 in the conventional pathway,our proposed FNA nanoplatform shows a remarkable enhancement on AS 1411 transport into cancer cells,with an implication for regulating cell signals and improving cancer targetability via environmental recognition.This thesis makes full use of the physical and chemical properties of DNA molecules to construct multiple DNA molecular devices,and apply them to analytical sensing,cell imaging,anti-cancer drug delivery and other fields.We can design and manipulate DNA molecular devices for molecular sensing,logic operations,disease diagnosis and other fields.In the next step,we hope to develop multi-responsive DNA molecular devices that can respond to multiple stimuli based on previous researches,promoting the application of DNA molecular devices in more complex systems.They play important roles in the field of life sciences.