Intracellular Entropy‐Driven Multi‐Bit DNA Computing for Tumor Progression Discrimination

Tumor progressions such as metastasis are complicated events that involve abnormal expression of different miRNAs and enzymes. Monitoring these biomolecules in live cells with computational DNA nanotechnology may enable discrimination of tumor progression via digital outputs. Herein, we report intracellular entropy‐driven multivalent DNA circuits to implement multi‐bit computing for simultaneous analysis of intracellular telomerase and microRNAs including miR‐21 and miR‐31. These three biomolecules can trigger respective DNA strand displacement recycling reactions for signal amplification. They are visualized by fluorescence imaging, and their signal outputs are encoded as multi‐bit binary codes for different cell types. The results can discriminate non‐tumorigenic, malignant and metastatic breast cells as well as respective tumors. This DNA computing circuit is further performed in a microfluidic chip to differentiate rare co‐cultured cells, which holds a potential for the analysis of clinical samples.     

Bridging the Two Worlds: A Universal Interface between Enzymatic and DNA Computing Systems

Molecular computing based on enzymes or nucleic acids has attracted a great deal of attention due to the perspectives of controlling living systems in the way we control electronic computers. Enzyme‐based computational systems can respond to a great variety of small molecule inputs. They have the advantage of signal amplification and highly specific recognition. DNA computing systems are most often controlled by oligonucleotide inputs/outputs and are capable of sophisticated computing as well as controlling gene expressions. Here, we developed an interface that enables communication of otherwise incompatible nucleic‐acid and enzyme‐computational systems. The enzymatic system processes small molecules as inputs and produces NADH as an output. The NADH output triggers electrochemical release of an oligonucleotide, which is accepted by a DNA computational system as an input. This interface is universal because the enzymatic and DNA computing systems are independent of each other in composition and complexity.     

Nucleic Acid-to-Small Molecule Converter through Amplified Hairpin DNA Circuits

Many microRNAs (miRNAs) are characteristically found in cancer cells, making miRNAs promising marker biomolecules for cancer diagnosis and therapeutics. However, it is challenging to use miRNA as a cancer signature because it is difficult to convert the nucleic acid sequence information into molecular functionality. To address this challenge, we realize nucleic acid-to-small molecule converters using hairpin DNA circuits. Harnessing a Staudinger reduction as a trigger for the conversion, we constructed hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) circuits that respond to oncogenic miR-21. Fluorophore and dye molecules were released in response to miR-21 through the HCR, providing fluorogenic and chromogenic readouts. Selective cytotoxicity in miR-21-abundant cells was realized by the CHA to release the anticancer drug SN-38. This would be the first example of selective activation of a small-molecule prodrug triggered by oncogenic miRNA in human living cells.     

PAM-less conditional DNA substrates leverage trans-cleavage of CRISPR-Cas12a for versatile live-cell biosensing

The CRISPR-Cas system has been repurposed as a powerful live-cell imaging tool, but its utility is limited to genomic loci and mRNA imaging in living cells. Here, we demonstrated the potential of the CRISPR-Cas system as a generalizable live-cell biosensing tool by extending its applicability to monitor diverse intracellular biomolecules. In this work, we engineered a CRISPR-Cas12a system with a generalized stimulus-responsive switch mechanism based on PAM-less conditional DNA substrates (pcDNAs). The pcDNAs with stimulus-responsiveness toward a trigger were constructed from the DNA substrates featuring no requirement of a protospacer-adjacent motif (PAM) and a bubble structure. With further leveraging the trans-cleavage activity of CRISPR-Cas12a for signal reporting, we established a versatile CRISPR-based live-cell biosensing system. This system enabled the sensitive sensing of various intracellular biomolecules, such as telomerase, ATP, and microRNA-21, making it a helpful tool for basic biochemical research and disease diagnostics.

This work developed the PAM-less conditional DNA substrates that leverage the trans-cleavage effect of CRISPR-Cas12a to sense various biomolecules in living cells.     

A microchamber array for single cell isolation and analysis of intracellular biomolecules

We present a microfluidic device that enables the determination of intracellular biomolecules in multiple single cells. The cells are individually trapped and isolated in a microchamber array. Since the microchambers can be opened and closed reversibly, the cells can be exposed to different solutions sequentially, e.g. for incubation, washing steps, labelling and finally, for lysis. The tightly sealed microchambers enable the retention and analysis of cell lysate derived from single cells. The performance of the device is demonstrated by monitoring the levels of the cofactors NADPH and NADH both in healthy mammalian cells and in cells exposed to oxidative stress. The platform was also used to determine the toxic impact of the alkaloid camptothecin on the intracellular enzyme glucose-6-phosphate dehydrogenase levels. In general, the device is applicable for the analysis of cell auto-stimulation and the detection of intracellular metabolite concentration or expression levels of proteins.     

DNA Nanotechnology-based Biocomputing

With silicon-based microelectronic technology pushed to its limit,scientists hunt to exploit biomolecules to power the bio-computer as substitutes.As a typical biomolecule,DNA now has been employed as a tool to create computing systems because of its superior parallel computing ability and outstanding data storage capability.However,the key challenges in this area lie in the human intervention during the computation process and the lack of platforms for central processor.DNA nanotechnology has created hundreds of complex and hierarchical DNA nanostructures with highly controllable motions by exploiting the unparalleled self-recognition properties of DNA molecule.These DNA nanostructures can provide platforms for central processor and reduce the human intervention during the computation process,which can offer unprecedented opportunities for biocomputing.In this review,recent advances in DNA nanotechnology are briefly summarized and the newly emerging concept of biocomputing with DNA nanostructures is introduced.     

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.     

Methods of Split Reporter Reconstitution for the Analysis of Biomolecules

Intracellular signaling inside living cells is controlled by the specific localization of biomolecules, including proteins, with timescales ranging from milliseconds to several hours. To elucidate the related spatial and temporal signal processes, development of optical probes for cellular events is a challenging task in present studies. Herein, we describe recent advances in the basic design of the optical probes, which have been inspired by luminescent creatures, and their practical application to visualize intracellular events in living cells and animals. A discussion of different probe designs reveals their benefits and shortcomings.     

Evaluation of DNA Force Fields in Implicit Solvation

DNA structural deformations and dynamics are crucial to its interactions in the cell. Theoretical simulations are essential tools to explore the structure, dynamics, and thermodynamics of biomolecules in a systematic way. Molecular mechanics force fields for DNA have benefited from constant improvements during the last decades. Several studies have evaluated and compared available force fields when the solvent is modeled by explicit molecules. On the other hand, few systematic studies have assessed the quality of duplex DNA models when implicit solvation is employed. The interest of an implicit modeling of the solvent consists in the important gain in the simulation performance and conformational sampling speed. In this study, respective influences of the force field and the implicit solvation model choice on DNA simulation quality are evaluated. To this end, extensive implicit solvent duplex DNA simulations are performed, attempting to reach both conformational and sequence diversity convergence. Structural parameters are extracted from simulations and statistically compared to available experimental and explicit solvation simulation data. Our results quantitatively expose the respective strengths and weaknesses of the different DNA force fields and implicit solvation models studied. This work can lead to the suggestion of improvements to current DNA theoretical models.     

Functionalized Nanogap for DNA Read-Out: Nucleotide Rotation and Current-Voltage Curves

Functionalized nanogaps embedded in nanopores show a strong potential for enhancing the detection of biomolecules, their length, type, and sequence. This detection is strongly dependent on the features of the target biomolecules, as well as the characteristics of the sensing device. In this work, through quantum-mechanical calculations, we elaborate on representative such aspects for the specific case of DNA detection and read-out. These aspects include the influence of single DNA nucleotide rotation within the nanogap and the current-voltage (I-V) characteristics of the nanogap. The results unveil a distinct variation in the electronic current across the functionalized device for the four natural DNA nucleotides with the applied voltage. These also underline the asymmetric response of the rotating nucleotides on this applied voltage and the respective variation in the rectification ratio of the device. The electronic tunneling current across the nanogap can be further enhanced through the proper choice of an applied bias voltage. We were able to correlate the enhancement of this current to the nucleotide rotation dynamics and a shift of the electronic transmission peaks towards the Fermi level. This nucleotide specific shift further reveals the sensitivity of the device in reading-out the identity of the DNA nucleotides for all different configurations in the nanogap. We underline the important information that can be obtained from both the I-V curves and the rectification characteristics of the nanogap device in view of accurately reading-out the DNA information. We show that tuning the applied bias can enhance this detection and discuss the implications in view of novel functionalized nanopore sequencers.     

An electrochemical biosensor based on DNA “nano-bridge” for amplified detection of exosomal microRNAs

Exosomal miRNAs, as potential biomarkers in liquid biopsy for cancer early diagnosis, have aroused widespread concern. Herein, an electrochemical biosensor based on DNA “nano-bridge” was designed and applied to detect exosomal microRNA-21 (miR-21) derived from breast cancer cells. In brief, the target miR-21 can specifically open the hairpin probe 1(HP1) labeled on the gold electrode (GE) surface through strand displacement reaction. Thus the exposed loop region of HP1 can act as an initiator sequence to activate the hybridization chain reaction (HCR) between two kinetically trapped hairpin probes: HP2 immobilized on the GE surface and biotin labeled HP3 in solution. Cascade HCR leads to the formation of DNA “nano-bridge” tethered to the GE surface with a great deal of “piers”. Upon addition of avidin-modified horseradish peroxidase (HRP), numerous HRP were bound to the formed “nano-bridge” through biotin-avidin interaction to arouse tremendous current signal. In theory, only a single miR-21 is able to trigger the continuous HCR between HP2 and HP3 until all of the HP2 are exhausted. Therefore the proposed biosensor achieved ultrahigh sensitivity toward miR-21 with the detection limit down to 168 amol/L, as well as little cross-hybridization even at the single-base-mismatched level. Successful attempts were also made in the detection of exosomal miR-21 obtained from the MCF-7 of breast cancer cell line. To our knowledge, this is the first attempt to built horizontal DNA nano-structure on the electrode surface for exosomal miRNAs detection. In a word, the high sensitivity, selectivity, low cost make the proposed method hold great potential application for early point-of-care (POC) diagnostics of cancer.     

Raman imaging of single living cells: probing effects of non-cytotoxic doses of an anti-cancer drug

Identifying cell response to a chemotherapy drug treatment, in particular at the single cell level, is an important issue in patient management. This study aims at evaluating the effect of gemcitabine on single living cells using micro-Raman imaging. We used as a model the non-small lung cancer cell line, Calu-1, exposed to cytostatic doses (1 nM to 1 μM for 24 h and 48 h) of gemcitabine, an antitumor drug currently used in the treatment of lung cancer. Following drug treatment as a function of doses and incubation times, the Raman maps of single living cells were acquired. Cell biomolecules (DNA, RNA, and proteins) were chemically extracted and their spectral signatures used to evaluate their respective distribution in the cellular spectral information of control and treated cells. The quantification of these distributions reveals a significant effect of 100 nM gemcitabine at 48 h incubation (concomitant decrease of nucleic acids and increase of proteins). PCA analyses performed both on nuclear and extracted biomolecules spectra show a time-dependent effect of the drug. These promising results reveal that effects of subtoxic doses can be monitored at the single cell level highlighting the importance of such studies for clinical applications.     

Volume exclusion influences in spectral characteristics of DNA-amino acids complexes

Living cells contain a variety of bio molecules including nucleic acids, proteins, polysaccharides, and metabolites as well as other soluble and insoluble components. These bio molecules occupy a significant fraction (20–40%) of the cellular volume. The total concentration of biomolecules reaches 400 g l⁻¹, leading to a crowded intracellular environment referred to as molecular crowding. This causes changes in chemical potential strongly affecting the physicochemical properties in the cell environment. Hence understanding the effects of molecular crowding conditions on biomolecules and biomolecular complexes is of utmost importance. In the present study, interaction of DNA with various amino acids has been observed under the influence of molecular crowding agents of various molecular sizes to understand the changes in DNA characteristics with reference to binding parameters to various amino acids using fluorescence and FTIR spectra. These studies are expected to indicate the changes in the DNA-amino acid complexes with particular significance of effects of molecular crowding in response to volume exclusion effects.     

A Smart Deoxyribozyme-Programmable Catalytic DNA Circuit for High-Contrast MicroRNA Imaging

Synthetic catalytic DNA circuits have been recognized as a promising signal amplification toolbox for sensitive intracellular imaging, yet their selectivity and efficiency are always constrained by uncontrolled off-site signal leakage and inefficient on-site circuitry activation. Thus, the endogenously controllable on-site exposure/activation of DNA circuits is highly desirable for achieving the selective imaging of live cells. Herein, an endogenously activated DNAzyme strategy was facilely integrated with a catalytic DNA circuit for guiding the selective and efficient microRNA imaging in vivo. To prevent the off-site activation, the circuitry constitute was initially caged without sensing functions, which could be selectively liberated by DNAzyme amplifier to guarantee the high-contrast microRNA imaging in target cells. This intelligent on-site modulation strategy can tremendously expand these molecularly engineered circuits in biological systems.     

Probing spatial organization of DNA strands using enzyme-free hairpin assembly circuits

Catalyzed hairpin assembly (CHA) is a robust enzyme-free signal-amplification reaction that has a wide range of potential applications, especially in biosensing. Although most studies of the analytical applications of CHA have focused on the measurement of concentrations of biomolecules, we show here that CHA can also be used to probe the spatial organization of biomolecules such as single-stranded DNA. The basis of such detection is the fact that a DNA structure that brings a toehold and a branch-migration domain into close proximity can catalyze the CHA reaction. We quantitatively studied this phenomenon and applied it to the detection of domain reorganization that occurs during DNA self-assembly processes such as the hybridization chain reaction (HCR). We also show that CHA circuits can be designed to detect certain types of hybridization defects. This principle allowed us to develop a "signal on" assay that can simultaneously respond to multiple types of mutations in a DNA strand in one simple reaction, which is of great interest in genotyping and molecular diagnostics. These findings highlight the potential impacts of DNA circuitry on DNA nanotechnology and provide new tools for further development of these fields.     

Emerging optofluidic technologies for point-of-care genetic analysis systems: a review

This review describes recently emerging optical and microfluidic technologies suitable for point-of-care genetic analysis systems. Such systems must rapidly detect hundreds of mutations from biological samples with low DNA concentration. We review optical technologies delivering multiplex sensitivity and compatible with lab-on-chip integration for both tagged and non-tagged optical detection, identifying significant source and detector technology emerging from telecommunications technology. We highlight the potential for improved hybridization efficiency through careful microfluidic design and outline some novel enhancement approaches using target molecule confinement. Optimization of fluidic parameters such as flow rate, channel height and time facilitates enhanced hybridization efficiency and consequently detection performance as compared with conventional assay formats (e.g. microwell plates). We highlight lab-on-chip implementations with integrated microfluidic control for “sample-to-answer” systems where molecular biology protocols to realize detection of target DNA sequences from whole blood are required. We also review relevant technology approaches to optofluidic integration, and highlight the issue of biomolecule compatibility. Key areas in the development of an integrated optofluidic system for DNA hybridization are optical/fluidic integration and the impact on biomolecules immobilized within the system. A wide range of technology platforms have been advanced for detection, quantification and other forms of characterization of a range of biomolecules (e.g. RNA, DNA, protein and whole cell). Owing to the very different requirements for sample preparation, manipulation and detection of the different types of biomolecules, this review is focused primarily on DNA–DNA interactions in the context of point-of-care analysis systems.     

A Closed Chemobrionic System as a Biochemical Delivery Platform

Inorganic cells bearing calcium silicate membranes were prepared and resembled closed chemical gardens. It was demonstrated that these inorganic cells can successfully be loaded with natural products, proteins and plasmid DNA, and their cargo can be released in a controlled manner. These cells demonstrated the ability of chemical gardens to act as platforms for the sustained delivery of biomolecules and are expected to introduce chemical gardens in the field of biosciences.     

Modular multi-level circuits from immobilized DNA-based logic gates

One of the fundamental goals of molecular computing is to reproduce the tenets of digital logic, such as component modularity and hierarchical circuit design. An important step toward this goal is the creation of molecular logic gates that can be rationally wired into multi-level circuits. Here we report the design and functional characterization of a complete set of modular DNA-based Boolean logic gates (AND, OR, and AND-NOT) and further demonstrate their wiring into a three-level circuit that exhibits Boolean XOR (exclusive OR) function. The approach is based on solid-supported DNA logic gates that are designed to operate with single-stranded DNA inputs and outputs. Since the solution-phase serves as the communication medium between gates, circuit wiring can be achieved by designating the DNA output of one gate as the input to another. Solid-supported logic gates provide enhanced gate modularity versus solution-phase systems by significantly simplifying the task of choosing appropriate DNA input and output sequences used in the construction of multi-level circuits. The molecular logic gates and circuits reported here were characterized by coupling DNA outputs to a single-input REPORT gate and monitoring the resulting fluorescent output signals.     

Light‐Triggered Capture and Release of DNA and Proteins by Host–Guest Binding and Electrostatic Interaction

The development of an effective and general delivery method that can be applied to a large variety of structurally diverse biomolecules remains a bottleneck in modern drug therapy. Herein, we present a supramolecular system for the dynamic trapping and light‐stimulated release of both DNA and proteins. Self‐assembled ternary complexes act as nanoscale carriers, comprising vesicles of amphiphilic cyclodextrin, the target biomolecules and linker molecules with an azobenzene unit and a charged functionality. The non‐covalent linker binds to the cyclodextrin by host–guest complexation with the azobenzene. Proteins or DNA are then bound to the functionalized vesicles through multivalent electrostatic attraction. The photoresponse of the host–guest complex allows a light‐induced switch from the multivalent state that can bind the biomolecules to the low‐affinity state of the free linker, thereby providing external control over the cargo release. The major advantage of this delivery approach is the wide variety of targets that can be addressed by multivalent electrostatic interaction, which we demonstrate on four types of DNA and six different proteins.