An approach for state differentiation in nucleic acid circuits: Application to diagnostic DNA computing.

BACKGROUND: Differentiating between different states in nucleic acid circuits is crucial for various biological applications. One approach, there is a requirement for complicated sequential summation, which can be excessive for practical purposes. By selectively labeling biologically significant states, this study tackles the issue and presents a more cost-effective and streamlined solution. The challenge is to efficiently distinguish between different states in a nucleic acid circuit. RESULTS: An innovative method is introduced in this study to distinguish between states in a nucleic acid circuit, emphasizing the biologically relevant ones. The circuit comprises four DNA logic gates and two detection modules, one for determining fetal gender and the other for diagnosing X-linked genetic disorders. The primary module generates a G-quadruplex DNAzyme when activated by specific biomarkers, which leads to a distinct colorimetric signal. The secondary module responds to hemophilia and choroideremia biomarkers, generating one or two DNAzymes. The absence of female fetus indicators results in no DNAzyme or color change. The circuit can differentiate various fetal states by producing one to four active DNAzymes in response to male fetus biomarkers. A single-color solution for state differentiation is provided by this approach, which promises significant advancements in DNA computing and diagnostic applications. SIGNIFICANCE: The innovative approach used in this study to distinguish states in nucleic acid circuits holds great significance. By selectively labeling biologically relevant states, circuit design is simplified and complexity is reduced. This advancement enables cost-effective and efficient diagnostic applications and contributes to DNA computing, providing a valuable solution to a fundamental problem.

Improving DNA nanostructure stability: A review of the biomedical applications and approaches.

DNA's programmable, predictable, and precise self-assembly properties enable structural DNA nanotechnology. DNA nanostructures have a wide range of applications in drug delivery, bioimaging, biosensing, and theranostics. However, physiological conditions, including low cationic ions and the presence of nucleases in biological systems, can limit the efficacy of DNA nanostructures. Several strategies for stabilizing DNA nanostructures have been developed, including i) coating them with biomolecules or polymers, ii) chemical cross-linking of the DNA strands, and iii) modifications of the nucleotides and nucleic acids backbone. These methods significantly enhance the structural stability of DNA nanostructures and thus enable in vivo and in vitro applications. This study reviews the present perspective on the distinctive properties of the DNA molecule and explains various DNA nanostructures, their advantages, and their disadvantages. We provide a brief overview of the biomedical applications of DNA nanostructures and comprehensively discuss possible approaches to improve their biostability. Finally, the shortcomings and challenges of the current biostability approaches are examined.

Digital data storage on DNA tape using CRISPR base editors.

While the archival digital memory industry approaches its physical limits, the demand is significantly increasing, therefore alternatives emerge. Recent efforts have demonstrated DNA's enormous potential as a digital storage medium with superior information durability, capacity, and energy consumption. However, the majority of the proposed systems require on-demand de-novo DNA synthesis techniques that produce a large amount of toxic waste and therefore are not industrially scalable and environmentally friendly. Inspired by the architecture of semiconductor memory devices and recent developments in gene editing, we created a molecular digital data storage system called "DNA Mutational Overwriting Storage" (DMOS) that stores information by leveraging combinatorial, addressable, orthogonal, and independent in vitro CRISPR base-editing reactions to write data on a blank pool of greenly synthesized DNA tapes. As a proof of concept, this work illustrates writing and accurately reading of both a bitmap representation of our school's logo and the title of this study on the DNA tapes.

In-vitro validated methods for encoding digital data in deoxyribonucleic acid (DNA).

Deoxyribonucleic acid (DNA) is emerging as an alternative archival memory technology. Recent advancements in DNA synthesis and sequencing have both increased the capacity and decreased the cost of storing information in de novo synthesized DNA pools. In this survey, we review methods for translating digital data to and/or from DNA molecules. An emphasis is placed on methods which have been validated by storing and retrieving real-world data via in-vitro experiments.

An outlook on the current challenges and opportunities in DNA data storage.

Silicon is the gold standard for information storage systems. The exponential generation of digital information will exhaust the global supply of refined silicon. Therefore, investing in alternative information storage materials such as DNA has gained momentum. DNA as a memory material possesses several advantages over silicon-based data storage, including higher storage capacity, data retention, and lower operational energy. Routine DNA data storage approaches encode data into chemically synthesized nucleotide sequences. The scalability of DNA data storage depends on factors such as the cost and the generation of hazardous waste during DNA synthesis, latency of writing and reading, and limited rewriting capacity. Here, we review the current status of DNA data storage encoding, writing, storing, retrieving and reading, and discuss the technology's challenges and opportunities.

Digital data storage on DNA tape using CRISPR base editors.

While the archival digital memory industry approaches its physical limits, the demand is significantly increasing, therefore alternatives emerge. Recent efforts have demonstrated DNA's enormous potential as a digital storage medium with superior information durability, capacity, and energy consumption. However, the majority of the proposed systems require on-demand de-novo DNA synthesis techniques that produce a large amount of toxic waste and therefore are not industrially scalable and environmentally friendly. Inspired by the architecture of semiconductor memory devices and recent developments in gene editing, we created a molecular digital data storage system called "DNA Mutational Overwriting Storage" (DMOS) that stores information by leveraging combinatorial, addressable, orthogonal, and independent in vitro CRISPR base-editing reactions to write data on a blank pool of greenly synthesized DNA tapes. As a proof of concept, we wrote both a bitmap representation of our school's logo and the title of this study on the DNA tapes, and accurately recovered the stored data.

An alternative approach to nucleic acid memory.

DNA is a compelling alternative to non-volatile information storage technologies due to its information density, stability, and energy efficiency. Previous studies have used artificially synthesized DNA to store data and automated next-generation sequencing to read it back. Here, we report digital Nucleic Acid Memory (dNAM) for applications that require a limited amount of data to have high information density, redundancy, and copy number. In dNAM, data is encoded by selecting combinations of single-stranded DNA with (1) or without (0) docking-site domains. When self-assembled with scaffold DNA, staple strands form DNA origami breadboards. Information encoded into the breadboards is read by monitoring the binding of fluorescent imager probes using DNA-PAINT super-resolution microscopy. To enhance data retention, a multi-layer error correction scheme that combines fountain and bi-level parity codes is used. As a prototype, fifteen origami encoded with 'Data is in our DNA!\n' are analyzed. Each origami encodes unique data-droplet, index, orientation, and error-correction information. The error-correction algorithms fully recover the message when individual docking sites, or entire origami, are missing. Unlike other approaches to DNA-based data storage, reading dNAM does not require sequencing. As such, it offers an additional path to explore the advantages and disadvantages of DNA as an emerging memory material.

Supramolecular aptamer nano-constructs for receptor-mediated targeting and light-triggered release of chemotherapeutics into cancer cells.

Platforms for targeted drug-delivery must simultaneously exhibit serum stability, efficient directed cell internalization, and triggered drug release. Here, using lipid-mediated self-assembly of aptamers, we combine multiple structural motifs into a single nanoconstruct that targets hepatocyte growth factor receptor (cMet). The nanocarrier consists of lipidated versions of a cMet-binding aptamer and a separate lipidated GC-rich DNA hairpin motif loaded with intercalated doxorubicin. Multiple 2',6'-dimethylazobenzene moieties are incorporated into the doxorubicin-binding motif to trigger the release of the chemotherapeutics by photoisomerization. The lipidated DNA scaffolds self-assemble into spherical hybrid-nanoconstructs that specifically bind cMet. The combined features of the nanocarriers increase serum nuclease resistance, favor their import into cells presumably mediated by endocytosis, and allow selective photo-release of the chemotherapeutic into the targeted cells. cMet-expressing H1838 tumor cells specifically internalize drug-loaded nanoconstructs, and subsequent UV exposure enhances cell mortality. This modular approach thus paves the way for novel classes of powerful aptamer-based therapeutics.

Twisting of DNA Origami from Intercalators.

DNA nanostructures represent the confluence of materials science, computer science, biology, and engineering. As functional assemblies, they are capable of performing mechanical and chemical work. In this study, we demonstrate global twisting of DNA nanorails made from two DNA origami six-helix bundles. Twisting was controlled using ethidium bromide or SYBR Green I as model intercalators. Our findings demonstrate that DNA nanorails: (i) twist when subjected to intercalators and the amount of twisting is concentration dependent, and (ii) twisting saturates at elevated concentrations. This study provides insight into how complex DNA structures undergo conformational changes when exposed to intercalators and may be of relevance when exploring how intercalating drugs interact with condensed biological structures such as chromatin and chromosomes, as well as chromatin analogous gene expression devices.

CAGE: Chromatin Analogous Gene Expression.

Self-assembled nucleic acids perform biological, chemical, and mechanical work at the nanoscale. DNA-based molecular machines have been designed here to perform work by reacting with cancer-specific miRNA mimics and then regulating gene expression in vitro by tuning RNA polymerase activity. Because RNA production is topologically restrained, the machines demonstrate chromatin analogous gene expression (CAGE). With modular and tunable design features, CAGE has potential for molecular biology, synthetic biology, and personalized medicine applications.

Metrology of DNA arrays by super-resolution microscopy.

Recent results in the assembly of DNA into structures and arrays with nanoscale features and patterns have opened the possibility of using DNA for sub-10 nm lithographic patterning of semiconductor devices. Super-resolution microscopy is being actively developed for DNA-based imaging and is compatible with inline optical metrology techniques for high volume manufacturing. Here, we combine DNA tile assembly with state-dependent super-resolution microscopy to introduce crystal-PAINT as a novel approach for metrology of DNA arrays. Using this approach, we demonstrate optical imaging and characterization of DNA arrays revealing grain boundaries and the temperature dependence of array quality. For finite arrays, analysis of crystal-PAINT images provides further quantitative information of array properties. This metrology approach enables defect detection and classification and facilitates statistical analysis of self-assembled DNA nanostructures.

A Coding Scheme for Nucleic Acid Memory (NAM).

The global demand for digital data is projected to be greater than the supply of semiconductor grade silicon in 2040 [1]. When combined with the need to archive information [2], nucleic acids are being explored as an alternative memory material [1-7]. According to a recent study, the information density of nucleic acid memory (NAM) is 1000 times greater than flash memory and has the ability to last for hundreds of years [1]. Presented here is an algorithm for converting digital data into unique DNA sequences for glacial storage. Biologically inspired, our coding scheme maps hexadecimal characters to sequences of three DNA nucleotides. This mapping avoids repeating sequences and start codons, which could have adverse effects. We were able to encode and decode various file types without error.

Construction of a fuzzy and Boolean logic gates based on DNA.

Logic gates are devices that can perform logical operations by transforming a set of inputs into a predictable single detectable output. The hybridization properties, structure, and function of nucleic acids can be used to make DNA-based logic gates. These devices are important modules in molecular computing and biosensing. The ideal logic gate system should provide a wide selection of logical operations, and be integrable in multiple copies into more complex structures. Here we show the successful construction of a small DNA-based logic gate complex that produces fluorescent outputs corresponding to the operation of the six Boolean logic gates AND, NAND, OR, NOR, XOR, and XNOR. The logic gate complex is shown to work also when implemented in a three-dimensional DNA origami box structure, where it controlled the position of the lid in a closed or open position. Implementation of multiple microRNA sensitive DNA locks on one DNA origami box structure enabled fuzzy logical operation that allows biosensing of complex molecular signals. Integrating logic gates with DNA origami systems opens a vast avenue to applications in the fields of nanomedicine for diagnostics and therapeutics.

Construction of a 4 zeptoliters switchable 3D DNA box origami.

The DNA origami technique is a recently developed self-assembly method that allows construction of 3D objects at the nanoscale for various applications. In the current study we report the production of a 18 × 18 × 24 nm(3) hollow DNA box origami structure with a switchable lid. The structure was efficiently produced and characterized by atomic force microscopy, transmission electron microscopy, and Förster resonance energy transfer spectroscopy. The DNA box has a unique reclosing mechanism, which enables it to repeatedly open and close in response to a unique set of DNA keys. This DNA device can potentially be used for a broad range of applications such as controlling the function of single molecules, controlled drug delivery, and molecular computing.

Structural DNA nanotechnology: from design to applications.

The exploitation of DNA for the production of nanoscale architectures presents a young yet paradigm breaking approach, which addresses many of the barriers to the self-assembly of small molecules into highly-ordered nanostructures via construct addressability. There are two major methods to construct DNA nanostructures, and in the current review we will discuss the principles and some examples of applications of both the tile-based and DNA origami methods. The tile-based approach is an older method that provides a good tool to construct small and simple structures, usually with multiply repeated domains. In contrast, the origami method, at this time, would appear to be more appropriate for the construction of bigger, more sophisticated and exactly defined structures.