Spatially Organized Enzymes Drive Cofactor-Coupled Cascade Reactions

We report the construction of an artificial enzyme cascade based on the xylose metabolic pathway. Two enzymes, xylose reductase and xylitol dehydrogenase, were assembled at specific locations on DNA origami by using DNA-binding protein adaptors with systematic variations in the interenzyme distances and defined numbers of enzyme molecules. The reaction system, which localized the two enzymes in close proximity to facilitate transport of reaction intermediates, resulted in significantly higher yields of the conversion of xylose into xylulose through the intermediate xylitol with recycling of the cofactor NADH. Analysis of the initial reaction rate, regenerated amount of NADH, and simulation of the intermediates’ diffusion indicated that the intermediates diffused to the second enzyme by Brownian motion. The efficiency of the cascade reaction with the bimolecular transport of xylitol and NAD⁺ likely depends more on the interenzyme distance than that of the cascade reaction with unimolecular transport between two enzymes

Table_1_Classification of Multiple DNA Dyes Based on Inhibition Effects on Real-Time Loop-Mediated Isothermal Amplification (LAMP): Prospect for Point of Care Setting.docx

LAMP has received great interest and is widely utilized in life sciences for nucleic acid analysis. To monitor a real-time LAMP assay, a fluorescence DNA dye is an indispensable component and therefore the selection of a suitable dye for real-time LAMP is a need. To aid this selection, we investigated the inhibition effects of twenty-three DNA dyes on real-time LAMP. Threshold time (Tt) values of each real-time LAMP were determined and used as an indicator of the inhibition effect. Based on the inhibition effects, the dyes were classified into four groups: (1) non-inhibition effect, (2) medium inhibition effect, (3) high inhibition effect, and (4) very high inhibition effect. The signal to noise ratio (SNR) and the limit of detection (LOD) of the dyes in groups 1, 2, and 3 were further investigated, and possible inhibition mechanisms of the DNA dyes on the real-time LAMP are suggested and discussed. Furthermore, a comparison of SYTO 9 in different LAMP reactions and different systems is presented. Of the 23 dyes tested, SYTO 9, SYTO 82, SYTO 16, SYTO 13, and Miami Yellow were the best dyes with no inhibitory effect, low LOD and high SNR in the real-time LAMP reactions. The present classification of the dyes will simplify the selection of fluorescence dye for real-time LAMP assays in point of care setting.</p

Data_Sheet_1_A Sensitive, Specific and Simple Loop Mediated Isothermal Amplification Method for Rapid Detection of Campylobacter spp. in Broiler Production.docx

Campylobacteriosis is one of the most common foodborne diseases worldwide. Two Campylobacter species – C. jejuni and C. coli in poultry and poultry products are considered to be the main source of human campylobacteriosis. Therefore, studying Campylobacter status in poultry flocks is needed to prevent transmission of disease and reduce human risk, health cost, and economic losses. In this study, we adapted and used a Loop-Mediated Isothermal Amplification (LAMP) assay for specific, sensitive, simple and cost-effective rapid detection of C. jejuni and C. coli in the poultry production chain. Amplified LAMP products were detected using a small, low-cost portable commercial blue LED transilluminator and a direct visual detection strategy was demonstrated. By using optimized conditions for amplification a limit of detection (LOD) of 50 CFU/ml was achieved for testing of C. jejuni and C. coli in spiked chicken feces without enrichment. The method took 60–70 min from receiving the samples to the final results (including 30 min for amplification). The optimized LAMP showed a relative accuracy of 98.4%, a specificity of 97.9%, and a sensitivity of 100% in comparison to real-time PCR method. Cohen’s kappa index also showed an excellent agreement (0.94) between the two methods. The results showed that the method is specific, sensitive and is suitable to develop for rapid detection of Campylobacter spp. at poultry production.</p