DNA logic programming: From concept to construction

DNA programming, which is based on the principle of base complementary pairing and Boolean operations, exhibits organizational structures and algorithms similar to those observed in machine language. Consequently, the practical implementation of DNA logic programming can be achieved through the utilization of programming techniques, enabling the discrimination and output generation. In recent years, DNA programming has witnessed a convergence with disciplines, such as life sciences, medicine, and other interdisciplinary areas, thereby giving rise to an advanced research system that yields valuable insights. This development has paved the way for multidisciplinary cutting‐edge research. Furthermore, the successful transition from conceptualization to the practical implementation of DNA programming has been accomplished. This review summarizes the recent advances in DNA logic programming within the biomedical fields, specifically emphasizing the conceptualization and execution of DNA logic programming constructs. The benefits and obstacles associated with the adoption of DNA programming in cutting‐edge research areas are also highlighted.

5][16][17][18] In 1994, Adleman proposed that DNA possesses the potential to serve as a means for computationally addressing a small example of a standard problem within the field of computer science, marking the initial establishment of DNA computing. 19On this basis, Milan Stojanovic and colleagues integrated digital circuits with DNA computing, introducing the novel concept of DNA logic gates.Utilizing DNA constructs, they assembled basic logic gates and achieved multilevel gate cascades, ultimately paving the way for a universal method of DNA computation. 20In a collaborative effort, Okamoto, Tanaka, and Saito introduced and formalized the concept of DNA logic gates in 2004, firmly setting up the research framework for DNA logical computation. 21hrough the hybridization process between a singlestranded (ss) oligonucleotide and its Watson-Crick complement, DNA assembly allows for the efficient design and implementation of higher-dimensional logical operations.][24][25] This approach facilitates the attainment of enhanced convenience, increased throughput, and expanded storage capacity for code editing purposes. 26,27In this case, foundational Boolean functions are established, forming the basis for DNA logic gates, encompassing fundamental gates, such as AND, OR, and NOT, alongside more advanced gates like complex logic gates and multi-input/output logic gates. 28,29By combining these gates, intricate logic circuits can be created, enabling the efficient information processing and computational operations.Thus, the integration of base complementary pairing and Boolean algorithm in DNA logic gates effectively introduces the scope of computational science into the realm of biomedical research. 30,31n recent years, DNA nanotechnology has experienced significant growth, driving advancements in biomedical research with the goal of enhancing its efficacy in disease theranostics, prognostic evaluation, and associated methodologies. 32,33][36] Significantly, through the organization and integration of DNA logic gates, the creation of DNA programming facilitates complex logic analysis. 37DNA programming operates on various logic gates while retaining their independent functions, thereby increasing the synergy between different gates to enable the analysis, judgment, and computation of complex phenomena from multiple dimensions.Compared with the linear discriminative mechanism of DNA logic gates, DNA logic programming enables the organic combination of multiple independent DNA logic gates, leading to the formation of a multiple, nonlinear discrimination approach for complex and ambiguous events, with the output of this discrimination conveyed in a multisignal manner.This strategy enhances the breadth and depth of discrimination capabilities of DNA logic gates to a certain extent. 38,39Currently, extensive attention is being devoted to the exploration of DNA programming, which is revealing remarkable vitality and great potential for further development across various disciplines .
This review provides a comprehensive overview of the functional mechanisms, applications and current research progress of DNA programming in the field of medical laboratory testing.Meanwhile, it systematically elucidates the pertinent research findings in recent years, spanning various levels including fundamental research, clinical analysis, and interdisciplinary extensions.Finally, the bottlenecks that need to be solved are objectively reviewed, and the prospects for the development of DNA programming technology in the biomedical field are anticipated (Figure 1).

FUNDAMENTAL PRINCIPLES AND WORKING MECHANISMS OF DNA PROGRAMMING
As mentioned above, DNA programming is a combination of DNA logic gates based on the Boolean algorithm and the fundamental principle of base complementary pairing. 40Emerging from the integration of biological science and information science, the fundamental concept of DNA programming involves the conversion of input signals into logical-based digital outputs utilizing a series of biochemical reactions adhering to Boolean algorithms. 41Boolean functions represent a set of principles governing logical operations that include Boolean operators such as NOT, AND, and OR.These functions can be implemented on DNA strands by facilitating a specific binding and conditional substitution between separate ss DNA. 42,43For example, a hybridization reaction targets a DNA sequence by introducing a supporting ss DNA molecule, usually a primer or probe.When specific DNA fragments bind together, double-stranded (ds) DNA structures that are consequentially formed are referred to as "1."Conversely, when the binding of DNA fragments is specifically impeded, it is denoted as "0."By utilizing specific primers and target sequences, a logical differentiation can be achieved between various binary states (0s and 1s), which are analogous to Boolean functions.Of note, the design of the output signal is determined by the complementary nature of DNA. 44ased on the classification of input signals, DNA logic gates can be identified into two distinct categories: single-signal and multisignal input DNA logic gates.The analysis and determination of individual targets frequently employ single-signal modes, which primarily rely on "YES" and "NOT" gates. 45For instance, circuittunable DNA nanomaterials can change their structures through the competitive binding of pH-dependent substrates and self-assembled DNA tiles to a catalyst strand, enabling self-assembly.Under neutral conditions, the catalyst strand induces a "YES" message response from the self-assembled DNA tiles. 46Conversely, when in an acidic state, it prompts a "NOT" response mediated by the inhibitory Hoogsteen structure formed by the pHdependent substrates and catalyst strands. 47otably, DNA logic gates, specifically designed for single-target analysis, yield accurate and consistent signal outputs through selective recognition mechanisms.These mechanisms enable molecular-level discrimination based solely on a single factor. 48Such discrimination is accomplished via exclusive recognition of either all or none of the target molecules.However, disease biomarkers in clinical samples often exhibit considerable heterogeneity.During multitarget analysis, these markers commonly interfere with one another, seriously limiting the specificity of clinical assays. 49,50Consequently, developing multiple DNA logic gates that satisfy real-world clinical needs becomes crucial.In the context of multiple DNA logic gates, distinct ssDNA or dsDNA are typically used to represent diverse signals, thereby forming signal groups.These groups exploit self-cycling to amplify signals or perform quantitative analysis, and to determine the authenticity of a given event using different signal codes. 51In essence, signal discrimination by multiple DNA logic gates extends beyond binary representation using only 0 and 1.It incorporates various combinations of AND, OR, and NOT in series or parallel configurations, thereby facilitating precise discrimination of intricate samples and permitting quantitative analysis.Compared to a single signal-DNA logic gate, multiple counterparts substantially expand the scope and extent of sample processing, enhancing testing accuracy considerably. 52,53DNA programming is the strategy of combining multiple DNA logic gates to produce a logical discriminatory group.The toehold sequences, which are responsible for executing discrimination tasks, autonomously regulate the production of free sequences.This regulation prompts the sequential binding and resultant conformational alterations of several nucleic acid molecules. 54These unique molecular conformational adjustments subsequently act as activating elements for the succeeding discriminative logic gates, thereby initiating a cascading mechanism analogous to a domino effect.The distinct logic gates can be engineered to function in either a series or parallel arrangement, thereby facilitating a concatenated discriminative reaction. 55This strategy represents the design philosophy and construction principles for implementing DNA logic programming.Meanwhile, functions such as "INHIBIT," "XOR," "NAND," and "NOR" following the Boolean function, have been incorporated into DNA programming to accommodate the complex sample environment and logic requirements.The prevalent DNA programming languages are exhibited in Figure 2. 56

DNA PROGRAMMING IN BIOMEDICAL FIELD
Clinical tests involve procedures for the identification, amplification, and analysis of target analytes. 57In this context, DNA logic gates utilize DNA as target recognition elements, executing precise recognition and capture processes through base complementary pairing, aptamer-ligand binding, and molecular conformational changes. 58,59Signal enhancement can be achieved by mechanisms like strand replacement and strand amplification.The results are then effectively interpreted using Boolean logic, enabling the intelligent integration of the clinical test procedure and results.1][62] The design principles of DNA logic circuits are rooted in DNA logic gates, which are characterized by probe recognition, conformational changes, information transmission, and in situ amplification.Built upon this foundation, DNA logic circuits integrate multiple different logic gate signals to form a logical information cascade with synchronized output.Depending on the nature of the target analyte, DNA logic circuits can be categorized for the analysis and determination of genes, specific proteins, and specific target molecules. 63To date, the combination of DNA logic gates with recognition and analysis methods of extracellular vesicles enables demonstrated effectiveness of low-abundance vesicles.This facilitates targeted detection of specific biological targets during the early onset of diseases. 64

DNA logic programming for precise gene analysis and regulation
The conventional method for nucleic acid detection relies on the precise identification of specific fragments fol-lowed by sequencing. 65,66Nevertheless, the interpretation of results proves arduous when analyzing multiple targets simultaneously.In contrast, DNA programming aims to synchronize target sequences by leveraging the distinct characteristics of various gene expressions and has the potential to overcome the limitation.For example, cell-based DNA sensors were developed by modifying the natural receptor bacteria, Bacillus subtilis, to detect specific DNA sequences in its environment. 67These DNA sensors exhibited the ability to identify a diverse array of bacterial species including prevalent human pathogens with high specificity.In another example, an orthogonal fluorescent reporter gene was incorporated into the sensing mechanism, achieving multiplex detection of genomic DNA from various species in complex samples. 68Meanwhile, the development of self-assembling DNA nanodecoys capable of recognizing multiple short RNA targets provided a way to detect and diagnose various respiratory viruses simultaneously. 69This innovative approach facilitated the identification of viral nucleic acid targets from multiple respiratory viruses, thereby addressing the challenge posed by the indistinguishable nature of these viruses.Further, DNA programming was applied to gene mapping resolution, recognizing multiple pieces of genetic information through one-pot ligase-dependent reactions based on the complementary decoding nature of DNA, and interpreting implicit genetic maps into explicit decision reports. 70,71his technique greatly enhanced the readability of gene maps, integrating synchronized target site analysis and precision discrimination into map parsing, and provided the possibility for further precision drug therapies.
In recent years, the specific regulation of targeted genes has emerged as a prominent research area.Notable advancements have been made in the CRISPR/Cas system, a gene editing technology that has garnered considerable The typical promotes prompts of 0 and 1arragement of DNA logic programming.Reproduced with permission from Ref. [76].Elsevier, copyright 2023.
interest in the gene editing field. 72,73Consequently, the integration of the CRISPR/Cas system with DNA programming holds the potential to facilitate effective and precise control over targeted gene expression, ultimately augmenting the efficacy of gene therapeutics. 74,75In a recent study, Liu et al. proposed a novel detection method for printed circuit boards (PCBs) by utilizing biocomputational logic gate programming and coupling the outputs of CRISPR/Cas12a as binary values (0 and 1). 76Enzyme markers are used to depict the absorbance value signals generated by the outputs of YES, OR, AND, and INHIBIT logic gates for quantitative visualization, thereby improving the accuracy of detecting small molecule nucleic acids.In existing work, 2,3',5,5'-tetrachlorobiphenyl (PCB72) and 3,3',4,4'tetrachlorobiphenyl (PCB77) are utilized as the two inputs to construct a biological computing logic gate.This forms a logic circuit-based biosensing platform for the simultaneous detection of multiple tetrachlorobiphenyls, enabling real-time tracking and dynamic monitoring of hazardous substances in the environment (Figure 3).

Potential applications of DNA programming for drug development
The development of novel drugs invariably strives for characteristics such as high-throughput screening and precise, personalized treatment.This approach enables the customization of drug administration based on individual differences and drug tolerances, thus enhancing the effectiveness of drug treatments and minimizing adverse effects. 77DNA programming, characterized by its specificity, accuracy, and programmability, lends itself to this pursuit.By employing a rational approach to design and selecting input signals, logical functions, and output signals, it becomes feasible to tailor and assemble the programming system according to customized requirements. 78,79The customization of the logic programming system enables the implementation of personalized drug therapies, thereby facilitating individualized drug treatment (Figure 4). 80he integration of drugs with logic gate systems expedites the screening and assessment of drug effects, thereby catalyzing the drug development process.For instance, the utilization of CRISPR-based orthogonal therapies, coupled with genetic logic control and precursor drug-based chemotherapy, has demonstrated a substantial capacity to impede tumor cell resistance. 81Meanwhile, the utilization of particular DNA sequences facilitates the efficient loading and accurate administration of chemotherapeutic agents to specific cells, thereby initiating a process known as "precision guidance." 82,83This process relies on the specific recognition between oligonucleotides and the simultaneous delivery of anticancer drugs, resulting in targeted delivery to the desired cells.On the other hand, DNA self-assembly through logical programming can respond to intracellular ATP molecules in a targeted manner, resulting in conformational changes and "0" and "1" response mechanisms, creating the possibility for intelligent delivery of drug molecules. 84t should be noted that gene repair, which is based on DNA programming principles, represents a substantial prospect for furthering gene-based pharmaceutical development.Recent studies have shown that the recognition, capture, and regulation of target genes by small interfering RNAs (siRNAs) may trigger the opening of DNA "gates" and release signals in situ, resulting in accurate and sensitive imaging analysis of miRNA-21 in tumor cells, thus building a comprehensive nanoplatform for precise cancer diagnosis and efficient gene therapy. 85oreover, the integration of aptamers with DNA programming expands the scope of nucleic acid functionality to include interactions with proteins and other small molecules.For example, to address the fact that exosomes of breast cancer cells exhibit high expression of specific proteins, the Wang group developed aptamers and logical programming for specific recognition.They identified trace tumor exosomal proteins in pathological samples with high throughput and sensitivity, and simultaneously performed specificity analysis and comparison with 97% accuracy. 86ptamers represent a class of short-stranded nucleic acids or proteins that specifically bind to target molecules, and they can be used as recognition elements in biosensors, in association with DNA logic gates, to achieve highly sensitive and selective detection of specific biomolecules. 87,88esigning an aptamer requires meticulous attention to its binding specificity and affinity towards the target molecule, as well as its impact on the operation of the DNA logic gate, ultimately leading to the generation The principle of DNA origami-based nanoplatforms for synergistic cancer therapy involving PTT and chemotherapy against breast cancer in vitro and in vivo.Reproduced with permission from Ref. [80], open access. of a conclusive judgment signal. 89Similarly, the design of DNA programming demands careful consideration of its logic operation function, stability, and compatibility with the aptamer.In a typical example, constructing an "AND" logic gate utilizing two distinct aptamerbinding hybridization chain reactions (HCR) enables the achievement of double satisfaction.This approach effectively facilitates the identification and isolation of four distinct tumor cell subtypes from clinical samples that consist predominantly of similar cell populations.Based on that, the application of aptamers and DNA programming is employed to achieve a two-factor "AND" detection of tumor microenvironmental tissues. 90This approach involves identifying distinct receptor molecules at the tumor site, enabling localized detection of tumors.In a specific study, a programming system composed of a sequence of DNA logic gates, including YES, AND, XOR, NOR, among others, was employed to concurrently identify four distinct subtypes of breast cancer cells. 91Importantly, this approach effectively mitigates the occurrence of falsepositive outcomes, a common issue linked with similar markers found in different tumor subtypes.In conclusion, the application of corresponding probes and a DNA programming system aimed at diverse targets possesses the capacity to enhance the detection of intricate samples and yield outcomes that are more sensitive and precise.

DNA logic circuits and advanced programming strategies
Inspired by concatenated logic circuits, DNA programming systems-designed with dual target-independent detection exhibit increased precision in synergistic diagnosis.For instance, using a dual detection circuit to identify the tumor-specific expression of target genes significantly improves the accuracy of tumor gene detection. 92Therefore, designing cyclic circuits while amplifying signals utilizing DNA logic programming systems is a crucial way to enhance the detection sensitivity.Leveraging the mutually activated cascade cycles of the HCR and catalytic hairpin assembly (CHA), Zhang et al. developed a DNA machine capable of delicate logic manipulation and molecular recognition, known as a closed-loop circuit. 93ssisted by synergistically enhanced signal amplification, the closed-loop DNA machine can execute logical operations and generate significance signals, even from weak input signals.
For the precise detection of low-abundance targets in complex samples, the simple design of DNA logic circuits using self-stacking-based on the fundamental cascade hybridization reaction (CHR)-enables the exponential amplification of target genes, which emphasizes the specificity discrepancy between targets and non-targets (Figure 5). 94On the other hand, the circular circuit system, rooted in DNA programming, has shown an exogenous immune function.It can be used to target small molecule CpG drug delivery based on cellular immunogenicity regulation, providing a novel concept for the development of "point-to-point" drug delivery. 92Furthermore, the programmable DNA computing system is used to create a decoder that can anticipate the calculation of a specific genetic map and output the corresponding decoded response automatically in a matter of hours with accuracy on par with Sanger sequencing, offering a comprehensive solution for the interpretation of gene mapping in clinical settings. 95

Application of synthetic DNA logic storage systems
The implementation of DNA logical storage is predicated upon the fundamental principles derived from the molecular structure and inherent properties of DNA. 96,97formation storage can be achieved by encoding data into DNA sequences and subsequently producing corresponding DNA molecules via biochemical reactions.Simultaneously, the process of deciphering the DNA molecule's sequence enables the extraction of informational content. 98DNA programming methods underpin the writing and reading of information within this procedure.Hence, DNA programming facilitates the conversion between binary numbers using a predetermined arrangement of the four bases.To date, DNA logical storage can be used to store vast amounts of biological data, opening up new avenues for advancements in disease research and treatment. 99To optimize DNA storage, obtaining the distribution of binary numbers is crucial prior to information encoding.By doing so, the corresponding bases can be placed more efficiently, reducing the synthesis of redundant information. 100Ultimately, this leads to higher density, faster information reading, and reduced DNA storage costs.
The stability and preservation properties of DNA structures enable the storage of DNA data on cellulose paper.
This method can produce a data volume with a computational density of up to 15 TB/mm 3 , paving the way for rapid data readout and economical preservation. 101In another example, conventional nucleic acid amplification technology has been integrated with DNA data replication.This results in DNA data being able to self-replicate within thermoresponsive, semipermeable microcapsules, allowing for effective amplification and preservation of information in samples of low abundance. 102Additionally, nanomaterials, such as metal-organic frameworks, offer robust and dependable encapsulation and immobilization for the preservation, delivery, and reading of DNA media. 103Meanwhile, the two-dimensional conformation of DNA offers potential for preserving image data, realizing the two two-dimensional encoding effect of images and opening avenues for the integration biological systems with digital technology.

3.5
The construction and storage of information leveraging DNA programming DNA logic circuits and storage have emerged as prominent research areas in contemporary biotechnology.Utilizing DNA molecules, these fields aim to accomplish computational and storage functions through specific biochemical reactions.This novel approach to information processing offers potential for the development of future biological computers, and provides new opportunities in bioinformatics, biomedicine, and other related disciplines.DNA logic circuits are formed by integrating various DNA logic gates, utilizing DNA logic algorithm technology.These circuits exhibit circuit characteristics through logical discrimination, generating responses and feedbacks. 104ence, similar to DNA logic circuits, the logical operation within the entire DNA logic circuit adheres strictly to the principle of complementary base pairing, thereby achieving a self-propelled circuit functionality.The primary application of DNA logic circuits focuses on bioinformatics and biomedicine, where they are used to accomplish complex information processing tasks, such as pattern recognition, data mining, intelligent drug design, ultimately realizing the precise diagnosis and treatment of diseases.
Specifically, the process of DNA logical storage involves encoding binary data using DNA bases and established coding rules. 105This procedure involves transforming binary files into DNA sequences and utilizing highthroughput synthesis technology to synthesize these sequences for information storage.Subsequent reading of the stored data employs high-throughput sequencing technology, wherein the binary file information is recovered based on the original coding rules.Expectedly, the use of DNA offers a stable and high-density storage solution for vast quantities of data, thanks to the advantageous properties of DNA including its exceptional stability and biocompatibility.The information storage process is achieved by harnessing DNA as a technological medium.When compared with traditional electronic storage technologies, it becomes apparent that DNA logic storage offers superior storage density and an extended storage lifespan. 106herefore, it is reasonably expected that the advancement and refinement of DNA logic circuits and logical storage will significantly facilitate the future development and improvement of current technologies.

Conclusion and Outlook
Due to its stable structure, superior biocompatibility, modifiability, and easy quantification, DNA is optimally suited as a molecular probe for precise target identification and labeling.Consequently, biotechnology built on DNA logic programming is projected to integrate with computational science, artificial intelligence (AI), and big data science, thus materializing the powerful correlation between biomedicine and information technology.In comparison to conventional detection technology, DNA programming has exhibited following advantages: (1) Intelligent interpretation: A closed-loop system based on DNA programming can execute the entire detection process-from identification to amplification to interpretation-independent of human or computer intervention.It synchronizes target identification with data analysis and result interpretation, resulting in highly intelligent interpretation.Furthermore, the ability of DNA to be conveniently labeled with various fluorescent dyes and chromogenic substances offers unique signal output modes, thereby facilitating the achievement of intelligent reading that is multithreaded and multimodular.Therefore, the DNA programming medical detection system is capable of carrying out comprehensive detection of target molecules in low abundance samples, thereby providing a convenient and efficient "one-stop" detection solution.
(2) Functional modularity: Due to technical limitations, most detection methods currently employed in clinical settings face challenges in realizing real-time editing for different detection targets.In contrast, DNA molecules with various configurations and functions can accurately determine different types of target molecules according to the actual needs through the processes of continuous recognition, binding, metastructuring, decomposition, and reconstruction, providing a novel approach for the rapid and precise detection of disease biomarkers.
(3) Superior flexibility: The advent of CRISPR gene editing and DNA nanotechnology (e.g., DNAzymes, isothermal amplification technology, and aptamers) has led to rapid enrichment and development of composite reading systems based on the logical programming of DNA as well as intelligent storage systems.As research progresses, these systems will continue to expand in terms of detection range and functionality.
At present, the field of DNA programming grapples with challenges such as standardized threshold quantification, low sensitivity, poor stability, analysis complexity of various samples, and reliance on a single judgment method.These common issues, however, continually being addressed with the aim of improving the field.aided by a deep integration of DNA nanotechnology with other emerging technologies, as well as the rapid advancement of interdisciplinary research.This progress propels the application of DNA nanotechnology across various fields.
To sum up, DNA programming offers substantial benefits and has immense potential in advanced scientific domains.Therefore, it necessitates further research and development to comprehensively understand its unique features and optimize its practical application.

A U T H O R C O N T R I B U T I O N S
This paper was written by Yi Zhang, Ning Hu.Jiajie Xu and Zhen Wang guided the structural arrangement and clinical application investigation for this paper.All revisions were discussed by Yi Zhang and Ning Hu.All authors read and approved the final manuscript.

A C K N O W L E D G E M E N T S
The work was financially supported by the Research Project of National Natural Science Foundation of China (8217082371), the key Project of Science and Technology of Zhejiang Province in China (2022C03186), and the Key projects by the province of Zhejiang Medical and Health Science and Technology Project (WKJ-ZJ-2101).Template pictures in Figure 1 were obtained from Biorender software.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

F I G U R E 1
Summary of DNA logic programming in four main domains.The mentioned applications cover studies of gene edition (single nucleotide and gene fragment editing by CRISPR-Cas strategy), drug development (specific nucleic acid structure construction), DNA logic circuits establishment and DNA logic storage (DNA strand assemble and self-driven reaction by nucleotide programming).All adaptions are open access.

F I G U R E 2
Truth and discrimination of multisignal DNA logic gates and a schematic representation.Reproduced with permission from Ref.[56], Elsevier, copyright,2022.

F I G U R E 5
Design and performance of the OR and AND gates by single nucleic acid strand in DNA logic circuit.Reproduced with permission from Ref.[94], Springer Nature, copyright 2019.