Suchergebnisse

An undecamer oligonucleotide probe based on a pair of deoxythymidines flanked by several modified nucleotides is a specific and highly efficient biosensor for micrococcal nuclease (MNase), an endonuclease produced by Staphylococcus aureus. Herein, the interaction mode and cleavage process on such oligonucleotide probes are identified and described for the first time. Also, we designed truncated pentamer probes as the minimum-length substrates required for specific and efficient biosensing. By means of computational (virtual docking) and experimental (ultra-performance liquid chromatography-mass spectrometry and matrix-assisted laser desorption ionization time-of-flight) techniques, we perform a sequence/structure-activity relationship analysis, propose a catalytically active substrate-enzyme complex, and elucidate a novel two-step phosphodiester bond hydrolysis mechanism, identifying the cleavage sites and detecting and quantifying the resulting probe fragments. Our results unravel a picture of both the enzyme-biosensor complex and a two-step cleavage/biosensing mechanism, key to the rational oligonucleotide design process. ; Funding Agencies|Wallenberg Centre for Molecular Medicine (WCMM) Linkoping, Sweden; Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linkoping University (Faculty grant SFO-Mat-LiU) [2009-00971]; Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency [MDM-2017-0720]; industrial doctorate program (MINECO) [DI-16-08891]; program Torres Quevedo (MINECO) [PTQ-17-09382]

Salmonella is a leading source of bacterial foodborne illness in humans, causing gastroenteritis outbreaks with bacteraemia occurrences that can lead to clinical complications and death. Eggs, poultry and pig products are considered as the main carriers of the pathogenic Salmonella for humans. To prevent this relevant zoonosis, key changes in food safety regulations were undertaken to improve controls in the food production chain. Despite these measures, large outbreaks of salmonellosis were reported worldwide in the last decade. Thus, new strategies for Salmonella detection are a priority for both, food safety and public health authorities. Such detection systems should provide significant reduction in diagnostic time (hours) compared to the currently available methods (days). Herein, we report on the discovery and characterization of nucleic acid probes for the sensitive and specific detection of live Salmonella within less than 8 h of incubation. We are the first to postulate the nuclease activity derived from Salmonella as biomarker of infection and its utility to develop innovative detection strategies. Our results have shown the screening and identification of two oligonucleotide sequences (substrates) as the most promising probes for detecting Salmonella - Sal-3 and Sal-5. The detection limits for both probes were determined with the reference Salmonella Typhimurium (STM 1) and Salmonella Enteritidis (SE 1) cultures. Sal-3 has reported LOD values around 10(5) CFU mL(-1) for STM 1 and 10(4) CFU mL(-1) for SE 1, while Sal-5 proves to be a slightly better probe, with LODs of 10(4) CFU mL(-1) for STM 1 and 10(4) CFU mL(-1) for SE 1. Both selected probes have shown the capability to recognize 49 out of 51 different Salmonella serotypes tested in vitro and the most frequent serotypes in porcine mesenteric lymph nodes as a standard sample used in fattening-pig salmonellosis baseline studies. Notably, our results showed 100% correlation between nuclease detection and the PCR-InvA or ISO-6579 standard method, underlining the great potential of this innovative nucleic acids technology to be implemented as a rapid method for food safety testing. (C) 2018 Elsevier B.V. All rights reserved. ; Funding Agencies|MINNECO [PTQ-16-08414]; Centre for the Development of Industrial Technology [CDTI] [20161256]; Departamento de Industria, Energia e Innovacion of the Navarra government, Spain [2016 PT071, 2017 PT031]; Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linkoping University [2009-00971]; Knut and Alice Wallenberg foundation

Simple Summary A diagnostic biomarker for the detection of breast cancer remains an unmet clinical need despite decades of intensive research efforts. Herein, we describe, for the first time, the use of nuclease activity as a biomarker to discriminate between healthy and cancer biopsy samples. We have identified a panel of three nucleic acid probes able to target nucleases derived from breast cancer tumors with high sensitivity and specificity. These results are in good agreement with histopathological analysis as the diagnostic gold standard. Moreover, these findings support nuclease activity as a potential adjacent diagnostic tool and shed light on the use of nuclease activity as a detection biomarker in breast cancer. Breast cancer is one of the most common pathologies diagnosed in the clinical practice. Despite major advancements in diagnostic approaches, there is no widely accepted biomarker in the clinical practice that can diagnose breast malignancy. Confirmatory diagnosis still relies on the pathological assessment of tissue biopsies by expert pathologists. Thus, there is an unmet need for new types of biomarkers and novel platform technologies that can be easily and robustly integrated into the clinic and that can assist pathologists. Herein, we show that nuclease activity associated to malignant tumors can be used as a novel biomarker in breast cancer, which can be detected via specific degradation of nucleic acid probes. In this study we have identified a set of three chemically modified nucleic acid probes that can diagnose malignancy in biopsy samples with high accuracy (89%), sensitivity (82%) and specificity (94%). This work represents a breakthrough for the potential clinical use of nuclease activity as biomarker, which can be detected via nucleic acids probes, for the clinical diagnosis of malignancy in breast tissue biopsies. This platform technology could be readily implemented into the clinic as adjunct to histopathological diagnostic. ; Funding Agencies|Wallenberg Centre for Molecular Medicine (WCMM) Linkoping, Sweden; Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linkoping University (Faculty Grant SFO-Mat-LiU) [2009-00971]; program Torres Quevedo (MINECO); industrial doctorate program (MINECO)