Multicomponent DNAzyme Nanomachines: Structure, Applications, and Prospects.

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.

A DAMP-Based Assay for Rapid and Affordable Diagnosis of Bacterial Meningitis Agents: Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae.

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.

DNA Nanomachine (DNM) Biplex Assay for Differentiating Bacillus cereus Species.

Conventional methods for the detection and differentiation of Bacillus cereus group species have drawbacks mostly due to the complexity of genetic discrimination between the Bacillus cereus species. Here, we describe a simple and straightforward assay based on the detected unamplified bacterial 16S rRNA by DNA nanomachine (DNM). The assay uses a universal fluorescent reporter and four all-DNA binding fragments, three of which are responsible for "opening up" the folded rRNA while the fourth stand is responsible for detecting single nucleotide variation (SNV) with high selectivity. Binding of the DNM to 16S rRNA results in the formation of the 10-23 deoxyribozyme catalytic core that cleaves the fluorescent reporter and produces a signal, which is amplified over time due to catalytic turnover. This developed biplex assay enables the detection of B. thuringiensis 16S rRNA at fluorescein and B. mycoides at Cy5 channels with a limit of detection of 30 × 103 and 35 × 103 CFU/mL, respectively, after 1.5 h with a hands-on time of ~10 min. The new assay may simplify the analysis of biological RNA samples and might be useful for environmental monitoring as a simple and inexpensive alternative to amplification-based nucleic acid analysis. The DNM proposed here may become an advantageous tool for detecting SNV in clinically significant DNA or RNA samples and can easily differentiate SNV under broadly variable experimental conditions and without prior amplification.

Visual Detection of Stem-Loop Primer Amplification (SPA) Products without Denaturation Using Peroxidase-like DNA Machines (PxDM).

Rapid, inexpensive, and accurate determination of nucleic acids is a decisive factor in evaluating population's health and monitoring treatment at point-of-care (POC) settings. Testing systems with visual outputs can provide instrument-free signal detection. Isothermal amplification technologies can substitute conventional polymerase chain reaction (PCR) testing due to compatibility with the POC diagnostic. Here, we have visually detected DNA fragments obtained by stem-loop-primer-assisted isothermal amplification (SPA), but not those obtained by PCR or LAMP amplification using DNA nanomachines with peroxidase-like activity (PxDM) with sensitivity to a single nucleotide substitution. Compared to the diagnostics with conventional loop-mediated isothermal amplification (LAMP), the PxDM method produces no false positive signals with the non-specific amplification products. The study suggests that PxDM, in conjunction with SPA isothermal amplification, can become a valid platform for POC testing systems.

The Aß42 Peptide and IAPP Physically Interact in a Yeast-Based Assay.

Numerous studies have demonstrated that people with type 2 diabetes mellitus (associated with IAPP peptide aggregation) show an increased incidence of Alzheimer's disease (associated with Aß aggregation), but the mechanism responsible for this correlation is presently unknown. Here, we applied a yeast-based model to study the interactions of IAPP with PrP (associated with TSEs) and with the Aß42 peptide. We demonstrated that fluorescently tagged IAPP forms detergent-resistant aggregates in yeast cells. Using the FRET approach, we showed that IAPP and Aß aggregates co-localize and physically interact in yeast cells. We also showed that this interaction is specific and that there is no interaction between IAPP and PrP in the yeast system. Our data confirmed a direct physical interaction between IAPP and Aß42 aggregates in a living cell. Based on these findings, we hypothesize that this interaction may play a crucial role in seeding Aß42 aggregation in T2DM patients, thereby promoting the development of AD.

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition.

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.

Detection of Multiplex NASBA RNA Products Using Colorimetric Split G Quadruplex Probes.

Structural RNA is a challenging target for recognition by hybridization probes. This chapter addresses the recognition problem of RNA amplicons in samples obtained by multiplex nucleic acid sequence-based amplification (NASBA). The method describes the design of G-quadruplex binary (split) DNA peroxidase sensors that produces colorimetric signal upon recognition of NASBA amplicons.

DNA nanomachine for visual detection of structured RNA and double stranded DNA.

Visual detection of ssRNA and dsDNA amplicons was achieved at room temperature without the need for a probe-analyte annealing stage. This approach uses a DNA nanostructure equipped with two analyte-binding arms. Highly selective binding of the third arm leads to the formation of a G-quadruplex structure capable of changing the solution color.

Toward a Home Test for COVID-19 Diagnosis: DNA Machine for Amplification-Free SARS-CoV-2 Detection in Clinical Samples.

Nucleic acid-based detection of RNA viruses requires an annealing procedure to obtain RNA/probe or RNA/primer complexes for unwinding stable structures of folded viral RNA. In this study, we designed a protein-enzyme-free nano-construction, named four-armed DNA machine (4DNM), that requires neither an amplification stage nor a high-temperature annealing step for SARS-CoV-2 detection. It uses a binary deoxyribozyme (BiDz) sensor incorporated in a DNA nanostructure equipped with a total of four RNA-binding arms. Additional arms were found to improve the limit of detection at least 10-fold. The sensor distinguished SARS-CoV-2 from other respiratory viruses and correctly identified five positive and six negative clinical samples verified by quantitative polymerase chain reaction (RT-qPCR). The strategy reported here can be used for the detection of long natural RNA and can become a basis for a point-of-care or home diagnostic test.

Development of molecular tools for diagnosis of Alzheimer's disease that are based on detection of amyloidogenic proteins.

Alzheimer's disease (AD) is the most common form of dementia that usually occurs among older people. AD results from neuronal degeneration that leads to the cognitive impairment and death. AD is incurable, typically develops over the course of many years and is accompanied by a loss of functional autonomy, making a patient completely dependent on family members and/or healthcare workers. Critical features of AD are pathological polymerization of Aß peptide and microtubule-associated protein tau, accompanied by alterations of their conformations and resulting in accumulation of cross-ß fibrils (amyloids) in human brains. AD apparently progresses asymptomatically for years or even decades before the appearance of symptoms. Therefore, development of the early AD diagnosis at a pre-symptomatic stage is essential for potential therapies. This review is focused on current and potential molecular tools (including non-invasive methods) that are based on detection of amyloidogenic proteins and can be applicable to early diagnosis of AD.Abbreviations: Aß - amyloid-ß peptide; AßO - amyloid-ß oligomers; AD - Alzheimer's disease; ADRDA - Alzheimer's Disease and Related Disorders Association; APH1 - anterior pharynx defective 1; APP - amyloid precursor protein; BACE1 - ß-site APP-cleaving enzyme 1; BBB - brain blood barrier; CJD - Creutzfeldt-Jakob disease; CRM - certified reference material; CSF - cerebrospinal fluid; ELISA - enzyme-linked immunosorbent assay; FGD - 18F-fluorodesoxyglucose (2-deoxy-2-[18F]fluoro-D-glucose); IP-MS - immunoprecipitation-mass spectrometry assay; MCI - mild cognitive impairment; MDS - multimer detection system; MRI - magnetic resonance imaging; NIA-AA - National Institute on Ageing and Alzheimer's Association; NINCDS - National Institute of Neurological and Communicative Disorders and Stroke; PEN2 - presenilin enhancer 2; PET - positron emission tomography; PiB - Pittsburgh Compound B; PiB-SUVR - PIB standardized uptake value ratio; PMCA - Protein Misfolding Cycling Amplification; PrP - Prion Protein; P-tau - hyperphosphorylated tau protein; RMP - reference measurement procedure; RT-QuIC - real-time quaking-induced conversion; SiMoA - single-molecule array; ThT - thioflavin T; TSEs - Transmissible Spongiform Encephslopathies; T-tau - total tau protein.

Functional Mammalian Amyloids and Amyloid-Like Proteins.

Amyloids are highly ordered fibrous cross-ß protein aggregates that are notorious primarily because of association with a variety of incurable human and animal diseases (termed amyloidoses), including Alzheimer's disease (AD), Parkinson's disease (PD), type 2 diabetes (T2D), and prion diseases. Some amyloid-associated diseases, in particular T2D and AD, are widespread and affect hundreds of millions of people all over the world. However, recently it has become evident that many amyloids, termed "functional amyloids," are involved in various activities that are beneficial to organisms. Functional amyloids were discovered in diverse taxa, ranging from bacteria to mammals. These amyloids are involved in vital biological functions such as long-term memory, storage of peptide hormones and scaffolding melanin polymerization in animals, substrate attachment, and biofilm formation in bacteria and fungi, etc. Thus, amyloids undoubtedly are playing important roles in biological and pathological processes. This review is focused on functional amyloids in mammals and summarizes approaches used for identifying new potentially amyloidogenic proteins and domains.