Versatile logic devices based on programmable DNA-regulated silver-nanocluster signal transducers

A DNA-encoding strategy is reported for the programmable regulation of the fluorescence properties of silver nanoclusters (AgNCs). By taking advantage of the DNA-encoding strategy, aqueous AgNCs were used as signal transducers to convert DNA inputs into fluorescence outputs for the construction of various DNA-based logic gates (AND, OR, INHIBIT, XOR, NOR, XNOR, NAND, and a sequential logic gate). Moreover, a biomolecular keypad that was capable of constructing crossword puzzles was also fabricated. These AgNC-based logic systems showed several advantages, including a simple transducer-introduction strategy, universal design, and biocompatible operation. In addition, this proof of concept opens the door to a new generation of signal transducer materials and provides a general route to versatile biomolecular logic devices for practical applications.     

Peptide‐Stabilized,Fluorescent Silver Nanoclusters: Solid‐Phase Synthesis and Screening

Few‐atom silver nanoclusters (AgNCs) can exhibit strong fluorescence; however, they require ligands to prevent aggregation into larger nanoparticles. Fluorescent AgNCs in biopolymer scaffolds have so far mainly been synthesized in solution, and peptides have only found limited use compared to DNA. Herein, we demonstrate how solid‐phase methods can increase throughput dramatically in peptide ligand screening and in initial evaluation of fluorescence intensity and chemical stability of peptide‐stabilized AgNCs (P‐AgNCs). 9‐Fluorenylmethyloxycarbonyl (Fmoc) solid‐phase peptide synthesis on a hydroxymethyl‐benzoic acid (HMBA) polyethylene glycol polyacrylamide copolymer (PEGA) resin enabled on‐resin screening and evaluation of a peptide library, leading to identification of novel peptide‐stabilized, fluorescent AgNCs. Using systematic amino acid substitutions, we synthesized and screened a 144‐member library. This allowed us to evaluate the effect of length, charge, and Cys content in peptides used as ligands for AgNC stabilization. The results of this study will enable future spectroscopic studies of these peptide‐stabilized AgNCs for bioimaging and other applications.     

DNA-silver Nanocluster Binary Probes for Ratiometric Fluorescent Detection of HPV-related DNA

Herein, we described a ratiometric strategy based on "chameleon" DNA-silver nanoclusters( DNA-AgNCs) fluorescent binary probes. The strategy was applied to detect high-risk human papillomavirus( HPV) DNA sequences, HPV-16. First, DNA-AgNCs were synthesized by a simple reduction method. The obtained nanoprobes showed typical yellow and red fluorescence of AgNCs. Upon the addition of HPV-16 DNA, the yellow fluorescence of AgNCs was reduced greatly, whereas tlie red fluorescence of AgNCs was increased. The concentration of HPV-16 DNA in the samples was characterized by the ratio of fluorescence intensity at 570 and 630 nm. Tlie ratiometric nanoprobes showed good selectivity for HPV-16 DNA, and the detection limit was 2 ninol/L. In addition, the practical applicability of this strategy was demonstrated by analysing the HPV-16 DNA in hiunan serum, illustrating its potential promise for clinical diagnosis.     

Label‐Free Logic Modules and Two‐Layer Cascade Based on Stem‐Loop Probes Containing a G‐Quadruplex Domain

A simple, versatile, and label‐free DNA computing strategy was designed by using toehold‐mediated strand displacement and stem‐loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two‐layer logic cascade were constructed. The probes contain a G‐quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light‐up fluorescent signal of G‐quadruplex/NMM complex was used as the output readout. The inputs are the disease‐specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label‐free and modular strategy might be adapted in multi‐target diagnosis through DNA hybridization and aptamer‐target interaction.     

Bioelectronic Interface Connecting Reversible Logic Gates Based on Enzyme and DNA Reactions

It is believed that connecting biomolecular computation elements in complex networks of communicating molecules may eventually lead to a biocomputer that can be used for diagnostics and/or the cure of physiological and genetic disorders. Here, a bioelectronic interface based on biomolecule‐modified electrodes has been designed to bridge reversible enzymatic logic gates with reversible DNA‐based logic gates. The enzyme‐based Fredkin gate with three input and three output signals was connected to the DNA‐based Feynman gate with two input and two output signals—both representing logically reversible computing elements. In the reversible Fredkin gate, the routing of two data signals between two output channels was controlled by the control signal (third channel). The two data output signals generated by the Fredkin gate were directed toward two electrochemical flow cells, responding to the output signals by releasing DNA molecules that serve as the input signals for the next Feynman logic gate based on the DNA reacting cascade, producing, in turn, two final output signals. The Feynman gate operated as the controlled NOT gate (CNOT), where one of the input channels controlled a NOT operation on another channel. Both logic gates represented a highly sophisticated combination of input‐controlled signal‐routing logic operations, resulting in redirecting chemical signals in different channels and performing orchestrated computing processes. The biomolecular reaction cascade responsible for the signal processing was realized by moving the solution from one reacting cell to another, including the reacting flow cells and electrochemical flow cells, which were organized in a specific network mimicking electronic computing circuitries. The designed system represents the first example of high complexity biocomputing processes integrating enzyme and DNA reactions and performing logically reversible signal processing.     

Polymer‐Templated Perylene‐Probe Noncovalent Self‐Assembly: A New Strategy for Label‐Free Ultrasensitive Fluorescence Turn‐On Biosensing

A new label‐free fluorescence turn‐on strategy for highly sensitive biosensing has been developed. A negatively charged perylene probe was synthesized. Polycations could induce aggregation of the perylene probe through noncovalent interactions and the fluorescence of the probe’s monomer was efficiently quenched. Upon addition of a single‐stranded nucleic acid, competitive binding of the negatively charged nucleic acid (a polyanion) to the cationic polymer resulted in the release of a monomer and thus a turn‐on fluorescence signal was detected. Without the use of any amplification techniques, a detection limit of 2 pM DNA was obtained. Based on these results, an assay strategy for the highly sensitive detection of alkaline phosphatase (ALP) activity has been demonstrated. λ Exonuclease (λ exo) could degrade 5′‐phosphorylated single‐stranded DNA. However, when the DNA sample was treated with ALP, the phosphate functional group was removed by ALP and it could no longer be degraded by λ exo. Binding of the DNA to the perylene probe–polycation complex resulted in a turn‐on fluorescence signal, which could be used for sensing of ALP. The method is highly sensitive, a limit of detection as low as 0.02 mU mL^?1 ALP was obtained. Our method is simple, convenient, highly sensitive, and inexpensive.     

Multi‐Level Logic Gate Operation Based on Amplified Aptasensor Performance

Conventional electronic circuits can perform multi‐level logic operations; however, this capability is rarely realized by biological logic gates. In addition, the question of how to close the gap between biomolecular computation and silicon‐based electrical circuitry is still a key issue in the bioelectronics field. Here we explore a novel split aptamer‐based multi‐level logic gate built from INHIBIT and AND gates that performs a net XOR analysis, with electrochemical signal as output. Based on the aptamer–target interaction and a novel concept of electrochemical rectification, a relayed charge transfer occurs upon target binding between aptamer‐linked redox probes and solution‐phase probes, which amplifies the sensor signal and facilitates a straightforward and reliable diagnosis. This work reveals a new route for the design of bioelectronic logic circuits that can realize multi‐level logic operation, which has the potential to simplify an otherwise complex diagnosis to a “yes” or “no” decision.     

DNA‐Programmed Dynamic Assembly of Quantum Dots for Molecular Computation

Despite the widespread use of quantum dots (QDs) for biosensing and bioimaging, QD‐based bio‐interfaceable and reconfigurable molecular computing systems have not yet been realized. DNA‐programmed dynamic assembly of multi‐color QDs is presented for the construction of a new class of fluorescence resonance energy transfer (FRET)‐based QD computing systems. A complete set of seven elementary logic gates (OR, AND, NOR, NAND, INH, XOR, XNOR) are realized using a series of binary and ternary QD complexes operated by strand displacement reactions. The integration of different logic gates into a half‐adder circuit for molecular computation is also demonstrated. This strategy is quite versatile and straightforward for logical operations and would pave the way for QD‐biocomputing‐based intelligent molecular diagnostics.     

Label‐Free Detection of MicroRNA: Two‐Step Signal Enhancement with a Hairpin‐Probe‐Based Graphene Fluorescence Switch and Isothermal Amplification

A label‐free approach with multiple enhancement of the signal for microRNA detection has been introduced. The key idea of this strategy is achieved by taking advantage of a novel graphene oxide (GO)/intercalating dye based fluorescent hairpin probe (HP) and an isothermal polymerization reaction. In this paper, we used microRNA‐21 (mir‐21) as the target to examine the desirable properties of this assay. When the target, as a “trigger”, was hybridized with the HP and caused a conformation change, an efficient isothermal polymerization reaction was activated to achieve the first step of the “signal” amplification. After incubation with the platform of GO/intercalating dye, the formed complex of DNA interacted with the high‐affinity dye and then detached from the surface of the GO, a process that was accompanied by distinguishable fluorescence recovery. Further signal enhancement has been accomplished by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. Due to the efficient and multiple amplification steps, this approach exerted a substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir‐21 at attomole levels. Proof‐of‐concept evidence has been provided for the proposed cost‐effective strategy; thus, this strategy could expand the application of GO‐material‐based bioanalysis for nucleic acid studies.     

Metal‐Ion‐Triggered Exonuclease III Activity for the Construction of DNA Colorimetric Logic Gates

The logic system is obtained by using a series of double‐stranded (ds) DNA templates with mismatched base pairs (T–T or C–C) and ion‐modulated exonuclease III (Exo III) activity, in which the Exo III cofactors, Hg²⁺ and Ag⁺ ions, are used as inputs for the activation of the respective scission of Exo III based on the formation of T–Hg²⁺–T or C–Ag⁺–C base pairs. Additionally, two kinds of signal probes are utilized to transduce the logic operations. One is the two split G‐rich DNA strands that are used to design the OR, AND, INHIBIT, and XOR gates, whereas the other is the self‐assembled split G‐quadruplex structure to construct NOR, NAND, IMPLICATION, and XNOR operations based on DNA hybridization and strand displacement. In the presence of hemin, the split G‐quadruplex biocatalyzes the formation of a colored product, which is an output signal for the different logic gates. Thus, we have constructed a complete set of colorimetric DNA logic gates based on the Exo III and split G‐quadruplex for the first time. In addition, we are able to effortlessly recognize the logic output signals by the naked eye and their simplicity and cost‐effective design is the most apparent feature for the logic gates developed in this work.     

INHIBIT‐Inspired Two‐Output DNA Logic Gates Based on Surface‐Enhanced Raman Scattering

Herein, we presented a novel logic gate based on an INHIBITION gate that performs parallel readouts. Logic gates performing INHIBITION and YES/OR were constructed using surface‐enhanced Raman scattering as optical outputs for the first time. The strategy allowed for simultaneous reading of outputs in one tube. The applicability of this strategy has been successfully exemplified in the construction of half‐adder using the two‐output logic gates as reporting gates. This reporting strategy provides additional design flexibility for dynamic DNA devices.     

Logic‐Gate‐Actuated DNA‐Controlled Receptor Assembly for the Programmable Modulation of Cellular Signal Transduction

Programming cells to sense multiple inputs and activate cellular signal transduction cascades is of great interest. Although this goal has been achieved through the engineering of genetic circuits using synthetic biology tools, a nongenetic and generic approach remains highly demanded. Herein, we present an aptamer‐controlled logic receptor assembly for modulating cellular signal transduction. Aptamers were engineered as “robotic arms” to capture target receptors (c‐Met and CD71) and a DNA logic assembly functioned as a computer processor to handle multiple inputs. As a result, the DNA assembly brings c‐Met and CD71 into close proximity, thus interfering with the ligand–receptor interactions of c‐Met and inhibiting its functions. Using this principle, a set of logic gates was created that respond to DNA strands or light irradiation, modulating the c‐Met/HGF signal pathways. This simple modular design provides a robust chemical tool for modulating cellular signal transduction.     

Patterned immobilization of biomolecules by using ion irradiation‐induced graft polymerization

A new method for biomolecular patterning based on ion irradiation‐induced graft polymerization was demonstrated in this study. Ion irradiation on a polymer surface resulted in the formation of active species, which was further used for surface‐initiated graft polymerization of acrylic acid. The results of the grafting study revealed that the surface graft polymerization using 20 vol % of acrylic acid on the poly(tetrafluoroethylene) (PTFE) film irradiated at the fluence of 1 × 10¹⁵ ions/cm² for 12 h was the optimum graft polymerization condition to achieve the maximum grafting degree. The results of the fluorescence microscopy also revealed that the optimum fluence to achieve the maximum fluorescence intensity was 1 × 10¹⁵ ions/cm². The grafting of acrylic acid on the PTFE surfaces was confirmed by a fluorescence labeling method. The grafted PTFE films were used for the immobilization of amine‐functionalized p‐DNA, followed by hybridization with fluorescently tagged c‐DNA. Biotin‐amine was also immobilized on the acrylic acid grafted PTFE surfaces. Successful biotin‐specific binding of streptavidin further confirmed the potential of this strategy for patterning of various biomolecules. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 6124–6134, 2009     

An Exonuclease III‐Powered,On‐Particle Stochastic DNA Walker

DNA‐based machines have attracted rapidly growing interest owing to their potential in drug delivery, biocomputing, and diagnostic applications. Herein, we report a type of exonuclease III (Exo III)‐powered stochastic DNA walker that can autonomously move on a spherical nucleic acid (SNA)‐based 3D track. The motion is propelled by unidirectional Exo III digestion of hybridized DNA tracks in a burnt‐bridge mechanism. The operation of this Exo III‐propelled DNA walker was monitored in real time and at the single‐particle resolution using total internal reflection fluorescence microscopy (TIRF). We further interrogated the morphological effect of the 3D track on the nuclease activity, which suggested that the performance of the DNA walker was critically dependent upon the DNA density and the track conformation. Finally, we demonstrated potential bioanalytical applications of this SNA‐based stochastic DNA walker by exploiting movement‐triggered cascade signal amplification.     

An Exonuclease III‐Powered,On‐Particle Stochastic DNA Walker

DNA‐based machines have attracted rapidly growing interest owing to their potential in drug delivery, biocomputing, and diagnostic applications. Herein, we report a type of exonuclease III (Exo III)‐powered stochastic DNA walker that can autonomously move on a spherical nucleic acid (SNA)‐based 3D track. The motion is propelled by unidirectional Exo III digestion of hybridized DNA tracks in a burnt‐bridge mechanism. The operation of this Exo III‐propelled DNA walker was monitored in real time and at the single‐particle resolution using total internal reflection fluorescence microscopy (TIRF). We further interrogated the morphological effect of the 3D track on the nuclease activity, which suggested that the performance of the DNA walker was critically dependent upon the DNA density and the track conformation. Finally, we demonstrated potential bioanalytical applications of this SNA‐based stochastic DNA walker by exploiting movement‐triggered cascade signal amplification.     

A Reversible DNA Logic Gate Platform Operated by One‐ and Two‐Photon Excitations

We demonstrate the use of two different wavelength ranges of excitation light as inputs to remotely trigger the responses of the self‐assembled DNA devices (D‐OR). As an important feature of this device, the dependence of the readout fluorescent signals on the two external inputs, UV excitation for 1 min and/or near infrared irradiation (NIR) at 800 nm fs laser pulses, can mimic function of signal communication in OR logic gates. Their operations could be reset easily to its initial state. Furthermore, these DNA devices exhibit efficient cellular uptake, low cytotoxicity, and high bio‐stability in different cell lines. They are considered as the first example of a photo‐responsive DNA logic gate system, as well as a biocompatible, multi‐wavelength excited system in response to UV and NIR. This is an important step to explore the concept of photo‐responsive DNA‐based systems as versatile tools in DNA computing, display devices, optical communication, and biology.     

Signal Propagation in Multi‐Layer DNAzyme Cascades Using Structured Chimeric Substrates

Signal propagation through enzyme cascades is a critical component of information processing in cellular systems. Although such systems have potential as biomolecular computing tools, rational design of synthetic protein networks remains infeasible. DNA strands with catalytic activity (DNAzymes) are an attractive alternative, enabling rational cascade design through predictable base‐pair hybridization principles. Multi‐layered DNAzyme signaling and logic cascades are now reported. Signaling between DNAzymes was achieved using a structured chimeric substrate (SCS) that releases a downstream activator after cleavage by an upstream DNAzyme. The SCS can be activated by various upstream DNAzymes, can be coupled to DNA strand‐displacement devices, and is highly resistant to interference from background DNA. This work enables the rational design of synthetic DNAzyme regulatory networks, with potential applications in biomolecular computing, biodetection, and autonomous theranostics.     

Direct Measurement of Single‐Molecule DNA Hybridization Dynamics with Single‐Base Resolution

Herein, we report label‐free detection of single‐molecule DNA hybridization dynamics with single‐base resolution. By using an electronic circuit based on point‐decorated silicon nanowires as electrical probes, we directly record the folding/unfolding process of individual hairpin DNAs with sufficiently high signal‐to‐noise ratio and bandwidth. These measurements reveal two‐level current oscillations with strong temperature dependence, enabling us to determine the thermodynamic and kinetic properties of hairpin DNA hybridization. More importantly, successive, stepwise increases and decreases in device conductance at low temperature on a microsecond timescale are successfully observed, indicating a base‐by‐base unfolding/folding process. The process demonstrates a kinetic zipper model for DNA hybridization/dehybridization at the single base‐pair level. This measurement capability promises a label‐free single‐molecule approach to probe biomolecular interactions with fast dynamics.     

Light‐Harvesting Nanoparticle Probes for FRET‐Based Detection of Oligonucleotides with Single‐Molecule Sensitivity

Controlling the emission of bright luminescent nanoparticles by a single molecular recognition event remains a challenge in the design of ultrasensitive probes for biomolecules. Herein, we developed 20‐nm light‐harvesting nanoantenna particles, built of a tailor‐made hydrophobic charged polymer poly(ethyl methacrylate‐co‐methacrylic acid), encapsulating circa 1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87‐fold brighter than quantum dots QDots 605 in single‐particle microscopy (with 550‐nm excitation), these DNA‐functionalized nanoparticles exhibit over 50 % total FRET efficiency to a single hybridized FRET acceptor, a highly photostable dye (ATTO665), leading to circa 250‐fold signal amplification. The obtained FRET nanoprobes enable single‐molecule detection of short DNA and RNA sequences, encoding a cancer marker (survivin), and imaging single hybridization events by an epi‐fluorescence microscope with ultralow excitation irradiance close to that of ambient sunlight.     

A splicing model‐based DNA‐computing approach on microfluidic chip

DNA computing is a new computation form based on DNA biochemical reactions, which is mainly composed of sticker and splicing computation models. In this work, a microfluidic chip‐based approach was established for splicing model‐based DNA computing. A finite automaton with two input symbols (a, b) and three states (S0, S1, and S2) was applied in the pattern recognition for isosceles triangles. The DNA computation processes of automaton were realized through DNA digestion, ligation, DNA separation, and detection on the microfluidic chip. The established approach is efficient, controllable, and easy to integrate, which paves the way for the building of complete biomolecular computers in the future.