Cationic copolymer and crowding agent have a cooperative effect on a Na+-dependent DNAzyme.

DNAzymes are promising agents for theranostics and biosensors. Sodium dependent DNAzymes have been developed for sensing and imaging of Na+, but these DNAzymes have low catalytic activity. Herein, we demonstrate that a molecular crowded environment containing 10 to 40 wt% PEG enhanced the catalytic activity of a Na+-dependent DNAzyme, EtNa, although dextran did not. The cationic copolymer poly(L-lysine)-graft-poly(ethylene glycol) at 0.03 wt% (0.3 g L-1) enhanced the reaction rate of EtNa by 10-fold, which is similar to the acceleration induced by 15 wt% (150 g L-1) PEG. A cooperative impact of the copolymer and crowding agent was observed: the combination resulted in an impressive 46-fold acceleration effect. Thus, the use of a cationic copolymer and a crowding agent is a promising strategy to improve the activity of Na+-dependent DNAzyme-based nanomachines, biosensors, and theranostics, especially in environments lacking divalent metal ions.

Label-free detection of HPV mRNA with an artificial chaperone-enhanced MNAzyme (ACEzyme)-based electrochemical sensor.

Nucleic acid biosensors for point-of-care (POC) diagnostic applications are highly desirable. The ability to detect DNA and RNA in a simple, rapid, affordable and portable format leads to a range of important applications for early screening in the field of disease monitoring and management. Herein, we report the development of an isothermal, label-free electrochemical biosensor that was designed on the basis of target-driven MNAzyme cleavage activity. Hybridization with HPV mRNA, a model nucleic acid target, activated MNAzyme and initiated the cleavage of immobilized hairpin substrates, leading to changes in the electrochemical signal. Under optimal conditions, a detection limit of 2.6 pM was obtained with an incubation time of 60 min. Furthermore, an artificial chaperone-enhanced MNAzyme (ACEzyme) system was integrated to an electrochemical biosensor for the first time. The analytical performance of the biosensor was enhanced, and the detection time was significantly reduced by the addition of PLL-g-Dex, which exhibits nucleic acid chaperone-like activity. A detection limit of 0.88 pM was obtained with a threefold decrease in incubation time without prior amplification. The proposed biosensing platform shows the advantages of simple fabrication and operation, good selectivity in the presence of single-base mismatch, and excellent versatility in a complex mixture of total RNA. We believe that this isothermal, label-free, and protein-free nucleic acid analysis platform could provide foundations for the further development of a universal nucleic acid biosensing platform for clinical application.

Label free electrochemical DNA biosensor for COVID-19 diagnosis.

The COVID-19 pandemic has significantly increased the development of the development of point-of-care (POC) diagnostic tools because they can serve as useful tools for detecting and controlling spread of the disease. Most current methods require sophisticated laboratory instruments and specialists to provide reliable, cost-effective, specific, and sensitive POC testing for COVID-19 diagnosis. Here, a smartphone-assisted Sensit Smart potentiostat (PalmSens) was integrated with a paper-based electrochemical sensor to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A disposable paper-based device was fabricated, and the working electrode directly modified with a pyrrolidinyl peptide nucleic acid (acpcPNA) as the biological recognition element to capture the target complementary DNA (cDNA). In the presence of the target cDNA, hybridization with acpcPNA probe blocks the redox conversion of a redox reporter, leading to a decrease in electrochemical response correlating to SARS-CoV-2 concentration. Under optimal conditions, a linear range from 0.1 to 200 nM and a detection limit of 1.0 pM were obtained. The PNA-based electrochemical paper-based analytical device (PNA-based ePAD) offers high specificity toward SARS-CoV-2 N gene because of the highly selective PNA-DNA binding. The developed sensor was used for amplification-free SARS-CoV-2 detection in 10 nasopharyngeal swab samples (7 SARS-CoV-2 positive and 3 SARS-CoV-2 negative), giving a 100% agreement result with RT-PCR.

One-step isothermal RNA detection with LNA-modified MNAzymes chaperoned by cationic copolymer.

RNA detection permits early diagnosis of several infectious diseases and cancers, which prevent propagation of diseases and improve treatment efficacy. However, standard technique for RNA detection such as reverse transcription-quantitative polymerase chain reaction has complicated procedure and requires well-trained personnel and specialized lab equipment. These shortcomings limit the application for point-of-care analysis which is critical for rapid and effective disease management. The multicomponent nucleic acid enzymes (MNAzymes) are one of the promising biosensors for simple, isothermal and enzyme-free RNA detection. Herein, we demonstrate simple yet effective strategies that significantly enhance analytical performance of MNAzymes. The addition of the cationic copolymer and structural modification of MNAzyme significantly enhanced selectivity and activity of MNAzymes by 250 fold and 2,700 fold, respectively. The highly simplified RNA detection system achieved a detection limit of 73 fM target concentration without additional amplification. The robustness of MNAzyme in the presence of non-target RNA was also improved. Our finding opens up a route toward the development of an alternative rapid, sensitive, isothermal, and protein-free RNA diagnostic tool, which expected to be of great clinical significance.

Artificial chaperones: From materials designs to applications.

Biological macromolecules must fold into native structures to gain functional activities. In living cells, proteins called molecular chaperones mediate productive folding by preventing undesired interactions and aggregation and by facilitating refolding of misfolded macromolecules into their bioactive forms. Inspired by natural molecular chaperones, artificial chaperones that mimic some features of their biological counterparts have been designed. This review describes recent progress in the development of artificial chaperones and their promising applications in enhancing macromolecular assembly of proteins, polypeptides, and nucleic acids.

Cationic copolymer enhances 8-17 DNAzyme and MNAzyme activities.

DNAzymes are DNA molecules capable of catalytic activity. The catalytic core of DNAzymes can be separated and conjugated with target binding arms to create allosteric DNAzymes known as multi-component nucleic acid enzymes (MNAzymes). Two widely used DNAzymes are the 10-23 and the 8-17 DNAzymes. These DNAzymes differ in catalytic core structures, cleavage sites, and reactive metal ion cofactors. Previously we showed that the presence of a cationic comb-type polymer poly(l-lysine)-graft-dextran (PLL-g-Dex) improved activities of the 10-23 DNAzyme and MNAzyme by facilitating assembly of the catalytic complex. In this work, we demonstrate that PLL-g-Dex enhances activities of the 8-17 DNAzyme and MNAzyme; poly(allylamine)-graft-dextran and cationic homopolymers did not enhance activities. Metal ion and pH dependences were observed in the presence of PLL-g-Dex, suggesting that the cationic copolymer did not impede the interaction between the metal ion and the DNA-based enzymes. Thus, PLL-g-Dex has chaperone-like activity for DNAzymes and MNAzymes regardless of structures, cleavage sites, and cofactors.

Cationic copolymer-chaperoned short-armed 10-23 DNAzymes.

The cationic copolymer poly(L-lysine)-graft-dextran (PLL-g-Dex) has nucleic acid chaperone-like activity. The copolymer facilitates both DNA hybridization and strand exchange reactions. For these reasons, DNA-based enzyme (DNAzyme) activity is enhanced in the presence of copolymer. In this study, we evaluated activities of DNAzymes with substrate-binding arms (S-arms) of various lengths. The copolymer promoted DNAzyme reactivity and turnover efficacy, and, depending on S-arm length, maximally accelerated the reaction rate by 250-fold compared to the rate in the absence of copolymer. The copolymer permitted up to six nucleotides truncation of the S-arms having initial length of 10 and 11 nucleotides without loss of catalytic efficiency, enable tuning of the optimal temperature ranging from 30 to 55 °C. The approach might be useful for the development of DNAzyme systems targeting short or highly structured RNAs as well for improvement of DNAzyme-based nanomachines and biosensors.

Cationic copolymer-chaperoned DNAzyme sensor for microRNA detection.

Multi-component nucleic acid enzymes (MNAzymes) are allosteric deoxyribozymes that are activated upon binding of a specific nucleic acid effector. MNAzyme activity is limited due to an insufficient assembly of the MNAzyme and its turnover. In this work, we describe the successful improvement of MNAzyme reactivity and selectivity by addition of cationic copolymers, which exhibit nucleic acid chaperone-like activity. The copolymer allowed a 210-fold increase in signal activity and a 95-fold increase in the signal-to-background selectivity of MNAzymes constructed for microRNA (miRNA) detection. The selectivity of the MNAzyme for homologous miRNAs was demonstrated in a multiplex format in which isothermal reactions of two different MNAzymes were performed. In addition, the copolymer permitted miRNA detections even in the presence of a ribonuclease which is ubiquitous in environments, indicating the protective effect of the copolymer against ribonucleases.

Turning hydrophilic bacteria into biorenewable hydrophobic material with potential antimicrobial activity via interaction with chitosan.

Alteration of a bacteriocin-producing hydrophilic bacterium, Lactococcus lactis IO-1, into a hydrophobic material with potential antimicrobial activity using chitosan was investigated and compared with five other bacterial species with industrial importance. The negatively charged bacterial cells were neutralized by positively charged chitosan, resulting in a significant increase in the hydrophobicity of the bacterial cell surface. The largest Gram-positive B. megaterium ATCC 14581 showed a moderate response to chitosan while the smaller E. coli DH5α, L. lactis IO-1 and P. putida F1 exhibited a significant response to an increase in chitosan concentration. Because L. lactis IO-1 is a good source for natural peptide lantibiotic that is highly effective against several strains of food spoilage organisms and pathogens, hydrophobic material derived from L. lactis IO-1 and chitosan is a promising novel material with antimicrobial activity for the food and pharmaceutical industries.