Split Fragments Research Articles

Chemical-tag labeling of proteins using fully recombinant split inteins.

Chemical-tag labeling of proteins involving split inteins is an approach for the selective chemical modification of proteins without the requirement of any chemical synthesis to be performed. In a two-step protocol, a very short tag fused to a split intein auxiliary protein is first labeled in a bioconjugation reaction with a synthetic moiety either at its N-terminus (amine-tag) or at the side chain of an unnatural amino acid (click-tag). The labeled protein is then mixed with the protein of interest fused to the complementary intein fragment. In the resulting spontaneous protein trans-splicing reaction the split intein fragments remove themselves and ligate the tag to the protein of interest in a virtually traceless fashion. The reaction can be performed either using a purified protein of interest or to label a protein in the context of a living cell. All protein components are recombinantly expressed and all chemical reagents are commercially available.

Time-resolved evolution of coherent structures in turbulent channels: characterization of eddies and cascades

AbstractA novel approach to the study of the kinematics and dynamics of turbulent flows is presented. The method involves tracking in time coherent structures, and provides all of the information required to characterize eddies from birth to death. Spatially and temporally well-resolved DNSs of channel data at $\mathit{Re}_{{\it\tau}}=930{-}4200$ are used to analyse the evolution of three-dimensional sweeps, ejections (Lozano-Durán et al., J. Fluid Mech., vol. 694, 2012, pp. 100–130) and clusters of vortices (del Álamo et al., J. Fluid Mech., vol. 561, 2006, pp. 329–358). The results show that most of the eddies remain small and do not last for long times, but that some become large, attach to the wall and extend across the logarithmic layer. The latter are geometrically and temporally self-similar, with lifetimes proportional to their size (or distance from the wall), and their dynamics is controlled by the mean shear near their centre of gravity. They are responsible for most of the total momentum transfer. Their origin, eventual disappearance, and history are investigated and characterized, including their advection velocity at different wall distances and the temporal evolution of their size. Reinforcing previous results, the symmetry found between sweeps and ejections supports the idea that they are not independent structures, but different manifestations of larger quasi-streamwise rollers in which they are embedded. Spatially localized direct and inverse cascades are respectively associated with the splitting and merging of individual structures, as in the models of Richardson (Proc. R. Soc. Lond. A, vol. 97(686), 1920, pp. 354–373) or Obukhov (Izv. Akad. Nauk USSR, Ser. Geogr. Geofiz., vol. 5(4), 1941, pp. 453–466). It is found that the direct cascade predominates, but that both directions are roughly comparable. Most of the merged or split fragments have sizes of the order of a few Kolmogorov viscous units, but a substantial fraction of the growth and decay of the larger eddies is due to a self-similar inertial process in which eddies merge and split in fragments spanning a wide range of scales.

In vivo co-localization of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent manner.

Co-localization of biochemical processes plays a key role in the directional control of metabolic fluxes toward specific products in cells. Here, we employ in vivo scaffolds made of RNA that can bind engineered proteins fused to specific RNA binding domains. This allows proteins to be co-localized on RNA scaffolds inside living Escherichia coli. We assembled a library of eight aptamers and corresponding RNA binding domains fused to partial fragments of fluorescent proteins. New scaffold designs could co-localize split green fluorescent protein fragments to produce activity as measured by cell-based fluorescence. The scaffolds consisted of either single bivalent RNAs or RNAs designed to polymerize in one or two dimensions. The new scaffolds were used to increase metabolic output from a two-enzyme pentadecane production pathway that contains a fatty aldehyde intermediate, as well as three and four enzymes in the succinate production pathway. Pentadecane synthesis depended on the geometry of enzymes on the scaffold, as determined through systematic reorientation of the acyl-ACP reductase fusion by rotation via addition of base pairs to its cognate RNA aptamer. Together, these data suggest that intra-cellular scaffolding of enzymatic reactions may enhance the direct channeling of a variety of substrates.

Highly selective and sensitive electrochemical biosensor for ATP based on the dual strategy integrating the cofactor-dependent enzymatic ligation reaction with self-cleaving DNAzyme-amplified electrochemical detection

A dual strategy that combines the adenosine triphosphate (ATP)-dependent enzymatic ligation reaction with self-cleaving DNAzyme-amplified electrochemical detection is employed to construct the biosensor. In this design, the methylene blue-labeled hairpin-structured DNA was self-assembled onto a gold electrode surface to prepare the modified electrode through the interaction of Au–S bond. In the procedure of ATP-dependent ligation reaction, when the specific cofactor ATP was added, the two split oligonucleotide fragments of 8-17 DNAzyme were linked by T4 DNA ligase and then released to hybridize with the labeled hairpin-structured DNA substrate. The linked 8-17 DNAzyme catalyzes the cleavage of the hairpin-structured substrate by the addition of Zn2+, causing the methylene blue which contains high electrochemical activity to leave the surface of the gold electrode, therefore generating a dramatic decrease of electrochemical signal. The decrease of peak current was readily measured by square wave voltammetry and a relatively low detection limit (0.05nM) was obtained with a linear response range from 0.1 to 1000nM. By taking advantage of the highly specific cofactor dependence of the DNA ligation reaction, the proposed ligation-induced DNAzyme cascades demonstrate ultrahigh selectivity toward the target cofactor ATP. A catalytic and molecular beacons strategy is further adopted to amplify the electrochemical signal detection achieved by cycling and regenerating the 8-17 DNAzyme to realize enzymatic multiple turnover, thus one DNAzyme can catalyze the cleavage of several hairpin-structured substrates, which improves the sensitivity of the newly designed electrochemical sensing system.

Split distance: a key landscape metric shaping amphibian populations and communities in forest fragments

AbstractAimHabitat split isolates forest fragments where terrestrial adults of many amphibian species live from aquatic‐breeding sites where their larvae develop, causing population declines and shifts in community structure. In contrast to most fragmentation theoretical frameworks which point to the relevance of fragment area and isolation, a recent theoretical model predicts that split distance, defined as the shortest distance between a forest fragment edge and the nearby stream, should affect negatively the abundance and occurrence probability of populations in forest fragments and, consequently, their total abundance and species richness. The model further predicts that the quality of the matrix between the two habitats, called split matrix, will modulate such responses. Here, we provide the first test of these theoretical predictions.LocationAtlantic Forest, Brazil.MethodsSplit distance was estimated for all fragments of the Brazilian Atlantic Forest, a highly disturbed biodiversity hotspot. Then, we selected 23 forest fragments, within a 1166‐km2 landscape, in a design that insured orthogonality between split distance (0–2000 m) and fragment size (1.7–30 ha). Split matrix quality (SMQ) and proximity, a measure of the availability of forest around the focal fragment, were estimated and controlled for in the analyses. Using multiple regression analyses with control for spatial autocorrelation, and AIC multimodel inference, we evaluated the relative importance of split distance, SMQ, fragment size and proximity to determine amphibian community and population attributes.ResultsMost Atlantic Forest fragments were affected by habitat split. Consistent with the theoretical predictions, species richness, abundance and the occurrence probability of many species declined with split distance and increased with SMQ, even when controlling for fragment size and isolation.Main conclusionsSplit distance can be used as a surrogate for conservation value of forest fragments for aquatic‐breeding amphibians in landscape planning and restoration enterprises.

In vivo imaging of protein-protein and RNA-protein interactions using novel far-red fluorescence complementation systems.

Imaging of protein–protein and RNA–protein interactions in vivo, especially in live animals, is still challenging. Here we developed far-red mNeptune-based bimolecular fluorescence complementation (BiFC) and trimolecular fluorescence complementation (TriFC) systems with excitation and emission above 600 nm in the ‘tissue optical window’ for imaging of protein–protein and RNA–protein interactions in live cells and mice. The far-red mNeptune BiFC was first built by selecting appropriate split mNeptune fragments, and then the mNeptune-TriFC system was built based on the mNeptune-BiFC system. The newly constructed mNeptune BiFC and TriFC systems were verified as useful tools for imaging protein–protein and mRNA–protein interactions, respectively, in live cells and mice. We then used the new mNeptune-TriFC system to investigate the interactions between human polypyrimidine-tract-binding protein (PTB) and HIV-1 mRNA elements as PTB may participate in HIV mRNA processing in HIV activation from latency. An interaction between PTB and the 3′long terminal repeat region of HIV-1 mRNAs was found and imaged in live cells and mice, implying a role for PTB in regulating HIV-1 mRNA processing. The study provides new tools for in vivo imaging of RNA–protein and protein–protein interactions, and adds new insight into the mechanism of HIV-1 mRNA processing.

Label-free detection of adenosine based on fluorescence resonance energy transfer between fluorescent silica nanoparticles and unmodified gold nanoparticles

A sensitive and convenient strategy was developed for label-free assay of adenosine. The strategy adapted the fluorescence resonance energy transfer property between Rhodamine B doped fluorescent silica nanoparticles (SiNPs) and gold nanoparticles (AuNPs) to generate signal. The different affinities of AuNPs toward the unfolded and folded aptamers were employed for the signal transfer in the system. In the presence of adenosine, the split aptamer fragments react with adenosine to form a structured complex. The folded aptamer cannot be adsorbed on the surface of AuNPs, which induces the aggregation of AuNPs under high ionic concentration conditions, and the aggregation of AuNPs leads to the decrease of the quenching ability. Therefore, the fluorescence intensity of Rhodamine B doped fluorescent SiNPs increased along with the concentration of adenosine. Because of the highly specific recognition ability of the aptamer toward adenosine and the strong quenching ability of AuNPs, the proposed strategy demonstrated good selectivity and high sensitivity for the detection of adenosine. Under the optimum conditions in the experiments, a linear range from 98nM to 100μM was obtained with a detection limit of 45nM. As this strategy is convenient, practical and sensitive, it will provide a promising potential for label-free aptamer-based protein detection.

A carbon nanotubes based fluorescent aptasensor for highly sensitive detection of adenosine deaminase activity and inhibitor screening in natural extracts

A carbon nanotubes (CNTs) based fluorescent aptasensor was developed for adenosine deaminase (ADA) activity detection and inhibitor screening by using adenosine (AD) as the substrate. This sensing system consists of CNTs, AD, split anti-AD aptamer fragment and dye-labeled aptamer fragment. In the absence of ADA, two aptamer fragments bind simultaneously with AD to form an AD-aptamer complex. This AD-aptamer complex cannot adsorb onto CNTs, and has high fluorescence intensity. When ADA is introduced into this system, ADA can convert AD into inosine, which has not affinity to the split anti-AD aptamer fragment. Thus, the split anti-AD aptamer fragments were adsorbed onto CNTs via strong π–π stacking interactions, resulting in the quenching of the fluorescence of the dye-labeled aptamer fragment. The proposed aptasensor can detect ADA activity from 0.005 to 0.2U/mL with a low detection limit of 0.002U/mL. Moreover, it has been also demonstrated that this CNTs-based fluorescence aptasensor is suitable for ADA inhibitor screening from traditional Chinese medicine (TCM). Considering the superior sensitivity and specificity, the proposed CNTs-based fluorescent aptasensor can be expected to provide a simple, cost-effective and sensitive platform for the detection of ADA activity and screening of potential drugs.

Target-Induced Conjunction of Split Aptamer Fragments and Assembly with a Water-Soluble Conjugated Polymer for Improved Protein Detection

Rapid and sensitive detection of proteins is crucial to biomedical research as well as clinical diagnosis. However, so far, most detection methods rely on antibody-based assays and are usually laborious and time-consuming, with poor sensitivity. Herein, we developed a simple and sensitive fluorescence-based strategy for protein detection by using split aptamer fragments and a water-soluble polycationic polymer (poly{[9,9-bis(6'-(N,N,N-diethylmethylammonium)hexyl)-2,7-fluorenylene ethynylene]-alt-co-[2,5-bis(3'-(N,N,N-diethylmethylammonium)-1'-oxapropyl)-1,4-phenylene] tetraiodide} (PFEP)). The thrombin-binding DNA aptamer was split into two fragments for target recognition. The PFEP with high fluorescence emission was used as energy donor to amplify the signal of dye-labeled DNA probe. In the absence of target, three DNA/PFEP complexes were formed via strong electrostatic interactions, resulting in efficient Föster resonance energy transfer (FRET) between two fluorophores. While the presence of target induces a conjunction of two split aptamer fragments to form G-quadruplex, and subsequent assemble with PFEP leading to the formation of G-quadruplex/thrombin/PFEP complex. The distance between the PFEP and dye increased due to protein's large size, leading to a remarkable decrease of the FRET signal. Compared with the intact aptamer, the use of shorter split aptamer fragments increases the possibility of forming G-quadruplex upon target. Thus, the rate of change of FRET signal before and after the addition of target improved significantly and a higher sensitivity (limit of detection (LOD) = 2 nM) was obtained. This strategy is superior in that it is rapid, has low cost and homogeneous detection, and does not need heating to avoid an unfavorable secondary structure of DNA probe. With further efforts, this method could be extended to a universal way for simple and sensitive detection of a variety of biomolecules.

Liquefaction of wood in hot compressed water Part 2 – Modeling of particle dissolution

The dissolution of wood in subcritical water is analyzed in order to provide insight into the effects of the operating parameters on the product distribution. A comprehensive mathematical model for the decomposition of wood by both the hydrolysis and pyrolysis reaction pathways was developed based on a theoretical understanding of the physical and chemical processes wood undergoes under these conditions and on the results of a series of batch experiments. Due to limitations in the experimental setup, kinetic information needed to be adapted from the literature in order to match the experimental data. By analysis of the experimental products, elemental balances for carbon, hydrogen and oxygen were also implemented in the model. Thus, the model is able to predict the final product distribution and the solid residue composition at different operating conditions. The lack of kinetic data for the fast initial reactions limits the applicability of the model. The model is also able to calculate particle size distributions. Comparison with measured distributions suggests that the wood particles do not shrink uniformly but split off smaller fragments.

A novel pair of split venus fragments to detect protein-protein interactions by in vitro and in vivo bimolecular fluorescence complementation assays.

Protein-protein interactions are critical components of almost every cellular process. The bimolecular fluorescence complementation (BiFC) method has been used to detect protein-protein interactions in both living cells and cell-free systems. The BiFC method is based on the principle that a fluorescent protein is reassembled from its two complementary non-fluorescent fragments when an interaction occurs between two proteins, each one fused to each fragment. In vivo and in vitro BiFC assays, which use a new pair of split Venus fragments composed of VN210 (amino acids 1-210) and VC210 (amino acids 210-238), are useful tools to detect and quantify various protein-protein interactions (including the cofilin-actin and Ras-Raf interactions) with high specificity and low background fluorescence. Moreover, these assays can be applied to screen small-molecule inhibitors of protein-protein interactions.

A signal-on fluorescence biosensor for detection of adenosine triphosphate based on click chemistry

A novel signal-on fluorescence biosensor for detection of adenosine triphosphate (ATP) based on the target induced recombination of split aptamer fragments and click chemistry is proposed.

Bioluminescent Probes to Analyze Ligand-Induced Phosphatidylinositol 3,4,5-Trisphosphate Production with Split Luciferase Complementation

A lipid second messenger, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), is a signaling molecule that mediates central cellular events, such as growth, motility, and development by activating downstream proteins. Although functions of various PIP3 binding partners have been unveiled, the various roles of PIP3 have not been resolved thoroughly because of limitations of PIP3 analysis. Herein, we describe a novel method for the analysis of relative PIP3 amount based on spontaneous complementation of split luciferase fragments. An N-terminal fragment of a luciferase was located on the plasma membrane (LucN-pm). A C-terminal fragment of a luciferase fused with PIP3 binding units, pleckstrin homology domains (PHDs) of the general receptor for phosphoinositides 1 (GRP1), was expressed in cytosol (PP-LucC). In response to PIP3 production, PP-LucC was brought to the plasma membrane and colocalized with LucN-pm. The LucN-pm and PP-LucC reconstituted spontaneously to form an active luciferase, producing bioluminescence recovery. We obtained bioluminescence signals corresponding to relative PIP3 amounts successfully upon stimulation with an agonist. We also demonstrated that the probes were applied for a high-throughput screening format and for monitoring of PIP3 production on the plasma membrane by bioluminescence. This method enables further study of PIP3 and supports versatile applications related to the PIP3 amount.

Imaging of mRNA–Protein Interactions in Live Cells Using Novel mCherry Trimolecular Fluorescence Complementation Systems

Live cell imaging of mRNA-protein interactions makes it possible to study posttranscriptional processes of cellular and viral gene expression under physiological conditions. In this study, red color mCherry-based trimolecular fluorescence complementation (TriFC) systems were constructed as new tools for visualizing mRNA–protein interaction in living cells using split mCherry fragments and HIV REV-RRE and TAT-TAR peptide-RNA interaction pairs. The new mCherry TriFC systems were successfully used to image RNA–protein interactions such as that between influenza viral protein NS1 and the 5’ UTR of influenza viral mRNAs NS, M, and NP. Upon combination of an mCherry TriFC system with a Venus TriFC system, multiple mRNA–protein interactions could be detected simultaneously in the same cells. Then, the new mCherry TriFC system was used for imaging of interactions between influenza A virus mRNAs and some of adapter proteins in cellular TAP nuclear export pathway in live cells. Adapter proteins Aly and UAP56 were found to associate with three kinds of viral mRNAs. Another adapter protein, splicing factor 9G8, only interacted with intron-containing spliced M2 mRNA. Co-immunoprecipitation assays with influenza A virus-infected cells confirmed these interactions. This study provides long-wavelength-spectrum TriFC systems as new tools for visualizing RNA–protein interactions in live cells and help to understand the nuclear export mechanism of influenza A viral mRNAs.

Co-transcriptional production of RNA-DNA hybrids for simultaneous release of multiple split functionalities.

Control over the simultaneous delivery of different functionalities and their synchronized intracellular activation can greatly benefit the fields of RNA and DNA biomedical nanotechnologies and allow for the production of nanoparticles and various switching devices with controllable functions. We present a system of multiple split functionalities embedded in the cognate pairs of RNA–DNA hybrids which are programmed to recognize each other, re-associate and form a DNA duplex while also releasing the split RNA fragments which upon association regain their original functions. Simultaneous activation of three different functionalities (RNAi, Förster resonance energy transfer and RNA aptamer) confirmed by multiple in vitro and cell culture experiments prove the concept. To automate the design process, a novel computational tool that differentiates between the thermodynamic stabilities of RNA–RNA, RNA–DNA and DNA–DNA duplexes was developed. Moreover, here we demonstrate that besides being easily produced by annealing synthetic RNAs and DNAs, the individual hybrids carrying longer RNAs can be produced by RNA polymerase II-dependent transcription of single-stranded DNA templates.

Expression and purification of recombinant NRL-Hsp90α and Cdc37-CRL proteins for in vitro Hsp90/Cdc37 inhibitors screening

Hsp90 has emerged as a promising target for cancer treatment. Hsp90 interacts with co-chaperone Cdc37 to mediate the conformational maturation of its kinase client proteins. Screening small molecule inhibitors targeting Hsp90/Cdc37 might be a promising strategy for further cancer therapeutic. In order to establish a recombinant protein system, the novel cloning and purification of full-length human Hsp90α and Cdc37 from BL21 (DE3) Escherichia coli is described here. In this work, we cloned and expressed recombinant NRL-Hsp90α and Cdc37-CRL that represent the full-length human Hsp90α and Cdc37 fused with the split Renilla luciferase (RL) protein fragments.We also expressed the full-length RL protein as a control for inhibitors screening. Moreover, we confirmed that the interaction proteins were able to complement split luciferase fragments and show the RL activity when substrate was added. In comparison, two mutations NRL-Hsp90α (Q133A) and Cdc37 (R167A)-CRL retained only 20% of the complemented RL activities. Six small molecule compounds were tested using this recombinant system. Very interestingly, Sulforaphane, Withaferin A, Celastrol and EGCG all decreased the complemented NRL-Hsp90α/Cdc37-CRL activities in the concentration-dependent manner. In addition, neither Sulforaphane nor Withaferin A showed non-specific inhibition on full length RL activity. However, Celastrol and EGCG showed different RL inhibition levels. The other two compounds LBH-589 and 17-AAG showed neither NRL-Hsp90α/Cdc37-CRL nor RL inhibition activities. These results indicate that purified NRL-Hsp90α and Cdc37-CRL appeared as pure, stable and active conformation, and can be used as an in vitro bioluminescence system for Hsp90/Cdc37 inhibitors screening.

Structural Complementation of a Monomeric Ribosomal-Inactivating Protein

Exotoxin A (ETA) from the Gram-negative bacterium Pseudomonas aeruginosa is a ribosomal-inactivating protein (RIP) that acts by ADP-ribosylation of the post-translationally modified histidine, diphthamide, in elongation factor 2. Apoptotic cell death typically ensues upon intoxication. Distinct from toxins with multiple subunits, ETA is a single-chain protein with three functional domains. The catalytic domain (domain III, or PE3) of pseudomonal exotoxin A is a 213-residue monomer with extensive sequence and structural homology with the A chain of diphtheria toxin and the more recently-characterized cholix toxin from Vibrio cholerae. We are interested in developing split fragments of PE3 capable of structural complementation. Based on B-factors in the crystal structure of PE3, we have identified several disordered loops as candidates for severance, none of which are spatially close to the active site. We have produced and characterized fusion constructs each consisting of a split fragment and one subunit of a heterospecific, antiparallel coiled-coil. We report one such pair that remains inactive individually but undergoes complementation to yield a functionally active RIP in vitro and we are characterizing its biophysical properties. To our knowledge, this pair of split PE3 fragments represents the first example structural complementation of a monomeric protein toxin and may have significant biotechnological and therapeutic applications. Our ultimate goal is to engineer a cellular toxin with multiple specificities to be achieved by conditional co-expression inside a target cell.

Streamlined Expressed Protein Ligation Using Split Inteins

Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

TAT hitchhiker selection expanded to folding helpers, multimeric interactions and combinations with protein fragment complementation

The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric protein-protein interactions enable TAT-dependent translocation of the resistance marker TEM β-lactamase (βL). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and βL, (ii) dimeric or oligomeric protein-protein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the βL, respectively and (iii) heterotrimeric protein-protein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split βL fragments and TorA, respectively. The lactamase fragments were additionally engineered for improved activity and stability. Applicability was benchmarked with interaction partners of known affinity and multimerization whereby cellular fitness correlated well with biophysical protein properties. Ultimately, the HiT selection was employed to identify peptides, which specifically bind to leukemia- and melanoma-relevant target proteins (MITF and ETO) by coiled-coil or tetra-helix-bundle formation with high affinity. The various versions of TAT selection led to inhibiting peptides (iPEPs) of disease-promoting interactions and enabled so far difficult to achieve selections.

Enzyme-amplified electronic logic gates based on split/intactaptamers

A series of enzyme-amplified electronic logic gates (OR, AND, NOR, and NAND) for one-spot simultaneous monitoring of small molecules and proteins has been constructed at the molecular level. This simple but universal system is based on target-induced self-assembly of split aptamer fragments or target-induced conformational changes of intact aptamers. For the OR and AND logic operations, the split aptamer fragments were used as the molecular recognition components, while for the NOR and NAND logic operations, the intact aptamers were used as the molecular recognition components. Using ATP and thrombin as inputs, the split/intact aptamers as molecular recognition elements, biotin as a tracer, and SA–HR as a reporter molecule, the electronic logic operations can be easily realized by generating amplified current signals as outputs. The logic system is robust and can be applied to human serum samples with excellent selectivity. Importantly, the reversibility of these logic gates makes the electronic system feasible to perform the set–reset function. Our work not only provides a “smart” and flexible logic platform for ATP and thrombin sensing, but also can be expanded for other ligands assay, such as adenosine monophosphate (AMP), theophylline, and cocaine, by rationally splitting their aptamer sequences into two fragments.