Nucleic acids (NAs) are important components of living organisms responsible for the storage and transmission of hereditary information. They form complex structures that can self-assemble and bind to various biological molecules. DNAzymes are NAs capable of performing simple chemical reactions, which makes them potentially useful elements for creating DNA nanomachines with required functions. This review focuses on multicomponent DNA-based nanomachines, in particular on DNAzymes as their main functional elements, as well as on the structure of DNAzyme nanomachines and their application in the diagnostics and treatment of diseases. The article also discusses the advantages and disadvantages of DNAzyme-based nanomachines and prospects for their future applications. The review provides information about new technologies and the possibilities of using NAs in medicine.

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided by a double-stranded DNA fragment, a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application in nucleic acid detection assays.

The rapid and accurate detection of viral infections is of paramount importance, given their widespread impact across diverse demographics. Common viruses such as influenza, parainfluenza, rhinovirus, and adenovirus contribute significantly to respiratory illnesses. The pathogenic nature of certain viruses, characterized by rapid mutations and high transmissibility, underscores the urgent need for dynamic detection methodologies. Quantitative reverse transcription PCR (RT-qPCR) remains the gold-standard diagnostic tool. Its reliance on costly equipment, reagents, and skilled personnel has driven explorations of alternative approaches, such as catalytic DNA nanomachines. Diagnostic platforms using catalytic DNA nanomachines offer amplification-free nucleic acid detection without the need for protein enzymes and demonstrate feasibility and cost-effectiveness for both laboratory and point-of-care diagnostics. This study focuses on the development of multicomponent DNA nanomachines with catalytic proficiency towards a fluorescent substrate, enabling the generation of a fluorescent signal upon the presence of target nucleic acids. Specifically tailored variants are designed for detecting human parainfluenza virus type 3 (HPIV) and respiratory syncytial virus (RSV). The engineered DNA nanomachine features six RNA-binding arms for recognition and unwinding of RNA secondary structures, along with a catalytic core for nucleic acid cleavage, indicating potential utility in real clinical practice with minimal requirements. This research showcases the potential of DNA nanomachines as a reliable and sensitive diagnostic tool for RNA virus identification, offering promising prospects for clinical applications in the realm of infectious disease management.

Objective: The 2020 pandemic showed a need for quick, accessible, and user-friendly diagnostic tests to detect various pathogens applicable at the point-of-care (POC) or at home. Such diagnostics should require a minimum of steps and equipment. Results and Discussion: In this study, we have manufactured and modified a one-way valve system for the microfluidic platform and integrate it to the device for DNA extraction and loop-mediated isothermal amplification (LAMP) with a color readout which allowed rapid and effective diagnosis of Neisseria meningitidis (N. meningitidis) and herpes simplex virus (HSV) without the use of expensive equipment. The article describes the aspects of the manufacturing process and a possible presentation of the platform. Conclusions: This article presents a solution of integrating of embedded flexible films with ion-dependent colorimetric visualization.

A multivalent 10–23 DNAzyme-based molecular nanomachine (DNM) enables sensitive, selective, and amplification-free detection of structured ssRNA and dsDNA.

The rapid and accurate diagnosis of meningitis is critical for preventing severe complications and fatalities. This study addresses the need for accessible diagnostics in the absence of specialized equipment by developing a novel diagnostic assay. The assay utilizes dual-priming isothermal amplification (DAMP) with unique internal primers to significantly reduce non-specificity. For fluorescence detection, the dye was selected among Brilliant Green, Thioflavin T, and dsGreen. Brilliant Green is preferred for this assay due to its availability, high fluorescence level, and optimal sample-to-background (S/B) ratio. The assay was developed for the detection of the primary causative agents of meningitis (Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae), and tested on clinical samples. The developed method demonstrated high specificity, no false positives, sensitivity comparable to that of loop-mediated isothermal amplification (LAMP), and a high S/B ratio. This versatile assay can be utilized as a standalone test or an integrated assay into point-of-care systems for rapid and reliable pathogen detection.