Primer exchange reaction-coupled transcription isothermal amplification as a sensitive biomolecular assay.

We devised a novel strategy that relies on a combination of the primer exchange reaction (PER) with transcription isothermal amplification, termed PER-Trap, for a sensitive biomolecular assay. Its design allowed light-up RNA aptamers to be produced as the final product, leading to the generation of an amplified fluorescence signal. The utility of PER-Trap was successfully demonstrated by the detection of exosomes.

Tetrahedral DNA nanostructures enhance transcription isothermal amplification for multiplex detection of non-coding RNAs.

This study introduces an innovative detection system for multiple cancer biomarkers, employing transcription isothermal amplification methods in conjunction with a tetrahedral DNA nanostructure (TDN). We demonstrate that TDN enhances various transcription isothermal amplification methods by placing DNA probes in proximity. Notably, the TDN-enhanced split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR) system stands out for its optimal performance and operational simplicity, especially in identifying non-coding RNAs such as microRNAs and long non-coding RNAs (lncRNAs). Multiplex detection of lncRNAs was also achieved by generating distinct light-up RNA aptamers, each emitting unique fluorescence signals. The system effectively identified the target lncRNAs, demonstrating high sensitivity and selectivity in both cell lines and clinical samples. The system, utilizing the single enzyme T7 RNA polymerase, can be easily tailored for alternative targets by substituting target-specific sequences in DNA probes and seamlessly integrated with other isothermal amplification methods for greater sensitivity and accuracy in the detection of multiple cancer biomarkers.

Aptamer-Based Switching System for Communication of Non-Interacting Proteins.

Biological macromolecules, such as DNA, RNA, and proteins in living organisms, form an intricate network that plays a key role in many biological processes. Many attempts have been made to build new networks by connecting non-communicable proteins with network mediators, especially using antibodies. In this study, we devised an aptamer-based switching system that enables communication between non-interacting proteins. As a proof of concept, two proteins, Cas13a and T7 RNA polymerase (T7 RNAP), were rationally connected using an aptamer that specifically binds to T7 RNAP. The proposed switching system can be modulated in both signal-on and signal-off manners and its responsiveness to the target activator can be controlled by adjusting the reaction time. This study paves the way for the expansion of biological networks by mediating interactions between proteins using aptamers.

Development of a Novel DNA Aptamer Targeting Colorectal Cancer Cell-Derived Small Extracellular Vesicles as a Potential Diagnostic and Therapeutic Agent.

Colorectal cancer (CRC) as the second leading cause of global cancer deaths poses critical challenges in clinical settings. Cancer-derived small extracellular vesicles (sEVs), which are secreted by cancer cells, have been shown to mediate tumor development, invasion, and even metastasis, and have thus received increasing attention for the development of cancer diagnostic or therapeutic platforms. In the present study, the sEV-targeted systematic evolution of ligands by exponential enrichment (E-SELEX) is developed to generate a high-quality aptamer (CCE-10F) that recognizes and binds to CRC-derived sEVs. Via an in-depth investigation, it is confirmed that this novel aptamer possesses high affinity (Kd = 3.41 nm) for CRC-derived sEVs and exhibits a wide linear range (2.0 × 104 -1.0 × 106 particles µL-1 ) with a limit of detection (LOD) of 1.0 × 103 particles µL-1 . Furthermore, the aptamer discriminates CRC cell-derived sEVs from those derived from normal colon cell, human serum, and other cancer cells, showing high specificity for CRC cell-derived sEVs and significantly suppresses the critical processes of metastasis, including cellular migration, invasion, and angiogenesis, which are originally induced by sEVs themselves. These findings are highly encouraging for the potential use of the aptamer in sEV-based diagnostic and therapeutic applications.

Cas12a/blocker DNA-based multiplex nucleic acid detection system for diagnosis of high-risk human papillomavirus infection.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins are an innovative tool in molecular diagnostics owing to their high specificity and modularity for target nucleic acid sequences. However, the sequence-indiscriminate trans-cleavage activity of the Cas protein renders multiplex detection challenging. In this study, we developed a Cas12a-based multiplex detection system by designing blocker DNA complementary to reporter DNA, which enables the simultaneous detection of two genes with a single Cas protein in a single reaction. As a proof of concept, we chose high-risk human papillomavirus (HPV) 16 and 18 as the model targets and incorporated recombinase polymerase amplification (RPA) and transcription reactions to achieve high accuracy and sensitivity. Using the proposed system, we detected the genes of both HPV 16 and 18 down to 1 aM within 80 min under isothermal conditions. We validated the performance of the system in detecting genomic DNA from various cell lines and clinical samples from cervical cancer patients with high specificity. The proposed system facilitated rapid multiplex detection of high-risk HPVs in a single reaction tube with only Cas12a, thus representing a more user-friendly and economical alternative to previous Cas protein-based multiplex detection assays. The proposed system has considerable potential for point-of-care testing and could be expanded to detect various nucleic acid biomarkers.

Low-Temperature Loop-Mediated Isothermal Amplification Operating at Physiological Temperature.

Loop-mediated isothermal amplification (LAMP) is one of the most widely used isothermal amplification technologies in molecular diagnostics. However, LAMP operates at a high temperature of 65 °C; thus, operating LAMP at a lower temperature is desirable to maximize its usefulness for on-site diagnosis. In this study, we propose a new version of LAMP, termed low-temperature LAMP, which operates at the physiological temperature of 37 °C. Low-temperature LAMP differs from conventional LAMP operating at 65 °C in terms of the concentrations of MgSO4 and deoxyribonucleoside triphosphates (dNTPs), as well as the lengths of DNA probes, which are crucial for the execution of low-temperature LAMP. Under the optimal conditions, the amplification efficiency of low-temperature LAMP is comparable to that of conventional LAMP. In addition, the ligation reaction at 37 °C, which is necessary to detect actual target nucleic acids, is combined without altering the temperature, enabling the identification of miR-21, a cancer-promoting oncogenic miRNA, with high sensitivity and selectivity. The method described in this paper does not require expensive DNA modifications or special additives and would facilitate the widespread application of LAMP in facility-limited or point-of-care settings, paving the way to improvements in other isothermal-amplification-based techniques.

Extracellular Vesicles, as Drug-Delivery Vehicles, Improve the Biological Activities of Astaxanthin.

Astaxanthin (AST) exhibits potent antioxidant and anti-inflammatory activities but poor stability and biological efficacy, which limit its application in the food and medical industries. In the present study, a new strategy was proposed to enhance the biological activities of AST using fetal bovine serum-derived extracellular vesicles (EVs). Saponin-assisted incubation was used to load AST owing to its high encapsulation efficiency and loading capacity. AST-incorporated EVs (EV-ASTs) maintained their original EV morphology and showed high stability at 4 °C, 25 °C, and 37 °C over a 28-day period, which was attributed to the protective environment provided by the phospholipid bilayer membrane of the EVs. Additionally, the EV-ASTs exhibited excellent antioxidant and anti-inflammatory activities in HaCaT keratinocytes and RAW 264.7 macrophage cells, respectively; these were significantly higher than those of free AST. Furthermore, the mechanism associated with the enhanced biological activities of EV-ASTs was evaluated by analyzing the expression of genes involved in antioxidation and anti-inflammation, in parallel with cellular in vitro assays. These results provide insights into methods for improving the performance of hydrophobic drugs using nature-derived EVs and will contribute to the development of novel drug-delivery systems.

Equipment-free, salt-mediated immobilization of nucleic acids for nucleic acid lateral flow assays.

As the world has been facing several deadly virus crises, including Zika virus disease, Ebola virus disease, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and Coronavirus disease 2019 (COVID-19), lateral flow assays (LFAs), which require minimal equipment for point-of-care of viral infectious diseases, are garnering much attention. Accordingly, there is an increasing demand to reduce the time and cost required for manufacturing LFAs. The current study introduces an equipment-free method of salt-mediated immobilization of nucleic acids (SAIoNs) for LFAs. Compared to general DNA immobilization methods such as streptavidin-biotin, UV-irradiation, and heat treatment, our method does not require special equipment (e.g., centrifuge, UV-crosslinker, heating device); therefore, it can be applied in a resource-limited environment with reduced production costs. The immobilization process was streamlined and completed within 30 min. Our method improved the color intensity signal approximately 14 times compared to the method without using SAIoNs and exhibited reproducibility with the long-term storage stability. The proposed method can be used to detect practical targets (e.g., SARS-CoV-2) and facilitates highly sensitive and selective detection of target nucleic acids with multiplexing capability and without any cross-reactivity. This novel immobilization strategy provides a basis for easily and inexpensively developing nucleic acid LFAs combined with various types of nucleic acid amplification.

DNA micelle-templated copper nanoclusters for fluorescent imaging of MUC1-positive cancer cells.

DNA micelles formed by hydrophobic, self-assembly of amphiphilic DNA monomers have enormous potential in biological imaging owing to its unique and programmable, three-dimensional nanostructure. Herein, we rationally design double-stranded DNA oligonucleotides with two cholesterols that can spontaneously form the lipid-mediated DNA micelles and generate the high fluorescence signal after the formation of DNA-templated copper nanoclusters (CuNCs). Furthermore, the DNA aptamer specific to MUC1 protein, aberrantly overexpressed on the surface of cancer cells, is attached to lipid-mediated DNA micelles to confer the selectivity towards the target cancer cells. With the well-defined DNA nanostructures, the cell membrane of MUC1-positive cancer cells are stained by CuNCs exhibiting an intense, red fluorescence signal, which are clearly distinguished from MUC1-negative cancer cells. This approach may not only expand the application scope of both DNA micells and CuNCs, especially in the area of cellular imaging, but also provides a basis for developing other types of DNA nanostructures to detect target biomarkers.

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor.

In addition to cis-cleavage activity that recognizes and cleaves nucleic acid sequences, a trans-cleavage activity that indiscriminately and non-specifically cleaves single-stranded DNA or RNA has been discovered in some Cas proteins, including Cas12a and Cas13a. Various detection methods using this activity have been widely reported. Herein, we describe a new highly efficient DNA reporter (5'-TTATT-CCCCC-3'; TTATT-5C) that outperformed the existing AT-rich DNA reporter (5'-TTATT-3') used in most Cas12a-based target nucleic detection assays. By systematically investigating the effect of DNA reporter length and sequence on the trans-cleavage activity of Cas12a, we achieved up to a 100-fold increase in fluorescence signal intensity derived from the trans-cleavage activity of Cas12a compared to that achieved using the existing AT-rich DNA reporter. The new DNA reporter was also applied, along with the existing AT-rich DNA reporter, for the detection of the Salmonella enterotoxin (stn) gene. Importantly, both detection speed and limit were significantly enhanced with the new DNA reporter. In addition, polymerase chain reaction (PCR) was adopted to the CRISR/Cas-Based system of the new DNA reporter, thereby confirming its practical applicability. The high-efficiency DNA reporter described herein can pave the way for further improving the trans-cleavage activity of other Cas proteins, as well as the sensitivity of CRISPR/Cas-Based systems. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-022-00081-0.

High Fructose Concentration Increases the Fluorescence Stability of DNA-Templated Copper Nanoclusters by Several Thousand Times.

DNA-templated copper nanoclusters (CuNCs) have limited applications because of their low fluorescence stability (several tens of minutes). In this study, we prepared CuNCs with improved temporal fluorescence stability by introducing fructose into the CuNC synthesis process and optimizing the reaction conditions. The inclusion of fructose increased the operating lifetime of CuNCs by approximately 5200-fold from 30 min to 108 days and improved their stability against heat, acids, and bases compared to CuNCs synthesized under original conditions. In addition, the fluorescence signal of CuNCs was maintained for a significantly longer time when stored at refrigeration (4 °C) and freezing (-20 °C) temperatures. Importantly, this method did not require the addition of substances other than fructose or any additional physicochemical treatment to maintain the fluorescence of DNA-templated CuNCs for more than several tens of days. As such, this study could serve as a basis to improve the stability of CuNCs for various applications.

Loop-mediated isothermal amplification-based nucleic acid lateral flow assay for the specific and multiplex detection of genetic markers.

In this study, a loop-mediated isothermal amplification-based nucleic acid lateral flow assay (LAMP-NALFA) system was developed for the specific and multiplex detection of genetic markers at a low cost. In principle, the LAMP reaction was optimized to generate a single-stranded sequence in the LAMP product, which was designed to serve as a barcode sequence and to specifically bind to the DNA capture on a NALFA strip. As a target genetic marker, the Salmonella enterotoxin (stn) gene was chosen and determined down to 9 aM (∼5.44 copies/µL). Importantly, the proposed system clearly discriminated the specific target amplification products from non-specific amplification products resulting from primers or non-target nucleic acids, proving the high selectivity of the LAMP-NALFA system. Furthermore, the practical applicability of the system was demonstrated by detecting Salmonella bacteria in Luria-Bertani medium, drinking water, and eggshells, with a limit of detection of 1.6 CFU. Finally, two different bacteria (Salmonella and Staphylococcus) were simultaneously determined by the multiplex LAMP-NALFA system. It is expected that the LAMP-NALFA system could be used in a point-of-care setting for the detection of bacteria or viruses, consequently improving both individual and public health.

DNA barcode-based detection of exosomal microRNAs using nucleic acid lateral flow assays for the diagnosis of colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths worldwide. The standard methods for diagnosing CRC, endoscopy and tissue biopsy, are invasive and time-consuming. Herein, we propose a novel method for the accurate and non-invasive diagnosis of CRC based on the analysis of exosomes that are circulating in biological fluids using a DNA barcode-based nucleic acid lateral flow assay (NALFA). Our technology combines reverse transcription using a stem-loop primer with DNA barcode-based NALFA. A colorimetric signal is generated only in the presence of the target exosomal miRNA, which can be determined even with the naked eye. The proposed system successfully detected miR-92a and miR-141, which are overexpressed in CRC exosomes. Moreover, when applied to plasma samples from CRC patients, our system simultaneously detected multiple markers in one strip. By combining these markers, we achieved high analytical performance with a sensitivity and a specificity of 95.24% and 100.0%, respectively, demonstrating that the proposed assay can be a simple diagnostic platform for the detection of exosomal miRNA.

T7 Endonuclease I-mediated voltammetric detection of KRAS mutation coupled with horseradish peroxidase for signal amplification.

Rapid and selective sensing of KRAS gene mutation which plays a crucial role in the development of colorectal, pancreatic, and lung cancers is of great significance in the early diagnosis of cancers. In the current study, we developed a simple electrochemical biosensor by differential pulse voltammetry technique for the specific detection of KRAS mutation that uses the mismatch-specific cleavage activity of T7-Endonuclease I (T7EI) coupled with horseradish peroxidase (HRP) to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) substrate in the presence of hydrogen peroxide (H2O2). In addition, we synthesized the nanocomposite composed of multi-walled carbon nanotube/chitosan-ionic liquid/gold nanoparticles (MWCNT/Chit-IL/AuNPs) on screen-printed carbon electrode surface to increase the electrode surface area and electrochemical signal. In principle, T7E1 enzyme recognized and cleaved the mismatched site formed by the presence of KRAS gene mutation, removing 5'-biotin of capture probes and subsequently reducing the differential pulse voltammetry signal compared to wild-type KRAS gene. With this proposed strategy, a limit of detection of 11.89 fM was achieved with a broad linear relationship from 100 fM to 1 µM and discriminated 0.1% of mutant genes from the wild-type target genes. This confirms that the developed biosensor is a potential platform for the detection of mutations in early disease diagnosis.

Three-Way Junction-Induced Isothermal Amplification with High Signal-to-Background Ratio for Detection of Pathogenic Bacteria.

The consumption of water and food contaminated by pathogens is a major cause of numerous diseases and deaths globally. To control pathogen contamination and reduce the risk of illness, a system is required that can quickly detect and monitor target pathogens. We developed a simple and reproducible strategy, termed three-way junction (3WJ)-induced transcription amplification, to detect target nucleic acids by rationally combining 3WJ-induced isothermal amplification with a light-up RNA aptamer. In principle, the presence of the target nucleic acid generates a large number of light-up RNA aptamers (Spinach aptamers) through strand displacement and transcription amplification for 2 h at 37 °C. The resulting Spinach RNA aptamers specifically bind to fluorogens such as 3,5-difluoro-4-hydroxybenzylidene imidazolinone and emit a highly enhanced fluorescence signal, which is clearly distinguished from the signal emitted in the absence of the target nucleic acid. With the proposed strategy, concentrations of target nucleic acids selected from the genome of Salmonellaenterica serovar Typhi (S. Typhi) were quantitatively determined with high selectivity. In addition, the practical applicability of the method was demonstrated by performing spike-and-recovery experiments with S. Typhi in human serum.

Synthesis of Exosome-Based Fluorescent Gold Nanoclusters for Cellular Imaging Applications.

In recent years, fluorescent metal nanoclusters have been used to develop bioimaging and sensing technology. Notably, protein-templated fluorescent gold nanoclusters (AuNCs) are attracting interest due to their excellent fluorescence properties and biocompatibility. Herein, we used an exosome template to synthesize AuNCs in an eco-friendly manner that required neither harsh conditions nor toxic chemicals. Specifically, we used a neutral (pH 7) and alkaline (pH 11.5) pH to synthesize two different exosome-based AuNCs (exo-AuNCs) with independent blue and red emission. Using field-emission scanning electron microscopy, energy dispersive X-ray microanalysis, nanoparticle tracking analysis, and X-ray photoelectron spectroscopy, we demonstrated that AuNCs were successfully formed in the exosomes. Red-emitting exo-AuNCs were found to have a larger Stokes shift and a stronger fluorescence intensity than the blue-emitting exo-AuNCs. Both exo-AuNCs were compatible with MCF-7 (human breast cancer), HeLa (human cervical cancer), and HT29 (human colon cancer) cells, although blue-emitting exo-AuNCs were cytotoxic at high concentrations (≥5 mg/mL). Red-emitting exo-AuNCs successfully stained the nucleus and were compatible with membrane-staining dyes. This is the first study to use exosomes to synthesize fluorescent nanomaterials for cellular imaging applications. As exosomes are naturally produced via secretion from almost all types of cell, the proposed method could serve as a strategy for low-cost production of versatile nanomaterials.

Fluorescence resonance energy transfer using DNA-templated copper nanoparticles for ratiometric detection of microRNAs.

We examined the effectiveness of a ratiometric method using DNA-templated copper nanoparticles, which can function as a probe for fluorescence resonance energy transfer. This method in combination with PCR successfully detected the target microRNA, which corresponded well with the results obtained by quantitative reverse transcription PCR.

Promoter engineering improves transcription efficiency in biomolecular assays.

We identified a novel 12 bp promoter that significantly increased transcription efficiency. Unlike the standard 20 bp promoter, which contains both recognition and initiation regions, the new promoter contains only a recognition region and is more suitable for diagnostic applications due to its smaller size. This promoter effectively produced different light-up RNA aptamers via transcription. Moreover, we used the promoter to analyze RNase H activity and achieved a detection limit of 0.009 U mL-1, which was significantly better than that achieved via previous methods. We propose that the new promoter may serve as a key component in various diagnostic applications.

Highly specific nuclear labeling via in situ formation of fluorescent copper nanoparticles.

When imaging cells, nuclear counterstaining is imperative; however, many commercial nuclear-staining dyes based on nucleic acid intercalation result in nonspecific signals in the cytoplasm. Here, we propose a new strategy that stains the nucleus with high specificity by in situ formation of DNA-templated copper nanoparticles (CuNPs). We demonstrated that genomic DNA in the nucleus enabled rapid formation of highly fluorescent CuNPs immediately following addition of a copper ion source and ascorbate as a reducing agent. Moreover, we found that RNA and mitochondrial DNA, largely responsible for nonspecific cytoplasmic signals from commercial nuclear-staining dyes, did not mediate the formation of the highly fluorescent CuNPs, resulting in highly specific nuclear staining at a reduced cost relative to commercially available methods. Furthermore, we verified the compatibility of the proposed method with other fluorescence-labeling techniques. These results demonstrated the efficacy of this method and its promise as a powerful tool for cell imaging.

Advances in Exosome Analysis Methods with an Emphasis on Electrochemistry.

Exosomes, small extracellular vesicles, are released by various cell types. They are found in bodily fluids, including blood, urine, serum, and saliva, and play essential roles in intercellular communication. Exosomes contain various biomarkers, such as nucleic acids and proteins, that reflect the status of their parent cells. Since they influence tumorigenesis and metastasis in cancer patients, exosomes are excellent noninvasive potential indicators for early cancer detection. Aptamers with specific binding properties have distinct advantages over antibodies, making them effective versatile bioreceptors for the detection of exosome biomarkers. Here, we review various aptamer-based exosome detection approaches based on signaling methods, such as fluorescence, colorimetry, and chemiluminescence, focusing on electrochemical strategies that are easier, cost-effective, and more sensitive than others. Further, we discuss the clinical applications of electrochemical exosome analysis strategies as well as future research directions in this field.