Stability and absorption mechanism of typical plant miRNAs in an in vitro gastrointestinal environment: basis for their cross-kingdom nutritional effects.

Plant miRNAs, a group of 19-24 nt noncoding RNAs from plant foods, were recently found to have immunomodulatory and nutritional effects on mammalian and human bodies. However, how the miRNAs survive gastrointestinal (GI) environment and how the stable miRNAs are absorbed, which serve the basis for their biological functions, were not unraveled. Here, we investigated the stabilities of six typical plant miRNAs in simulated gastric and intestinal environments, and the absorption mechanisms by Caco-2 cells. The results showed that the miRNAs can survive the environment with certain concentrations. The mixture of food ingredients enhanced the stabilities of the plant miRNAs in the gastric conditions, while 2'-O-methyl modification protects the miRNAs in intestinal juice. The stabilities of the miRNAs vary significantly in the environment and are related to their secondary structures. The stable plant miRNAs can be absorbed by Caco-2 cells via clathrin- and caveolin-mediated endocytosis. Uptake of the miRNAs was sequence dependent, facilitated by NACh and TLR9, two typical receptors on cell membrane. The results suggest that some of plant miRNAs are stable in the mimic GI environment and can be absorbed by Caco-2 cells, underlying the potential of their cross-kingdom regulation effects.

Rapid identification and quantitation of the viable cells of Lactobacillus casei in fermented dairy products using an aptamer-based strategy powered by a novel cell-SELEX protocol.

An aptamer-based strategy was developed for qualitative and quantitative analysis of viable Lactobacillus casei in dairy products. Three highly specific aptamers for L. casei were obtained using systematic evolution of ligands by exponential enrichment protocol using the whole bacterium cell as the target (cell-SELEX) facilitated by polyethyleneglycol and chitosan modified graphene oxide and complementary ring-mediated rolling circle amplification. Two aptamers, one for separating and enriching the L. casei cells and the other for generating fluorescence signals, were employed to develop an aptamer-based strategy, which was demonstrated for the selective detection of L. casei in commercial dairy drinks, with a dynamic range of 105 to 109 cfu/mL. Viable and nonviable L. casei cells could be discriminated based on the significant difference in fluorescence intensity. This established strategy is of high selectivity and sensitivity, and can be used for rapid analysis of viable L. casei in quality control and food surveillance areas.

A novel isothermal method using rolling circle reverse transcription for accurate amplification of small RNA sequences.

RNA amplification has extensive applications on biochemistry and its related fields. Here, we present an isothermal strategy named rolling circle reverse transcription-mediated RNA amplification (RCRT-MRA) to amplify small RNAs with accurate length and sequence. The target RNA and complementary DNA were circularized to serve as amplicons replicated via the rolling circle mechanism. The transcription product consisting of tandemly repeated RNA units, was monomerized by site-specific cleavage to generate amplified RNA with authentic length and sequence. T4 DNA ligase was chosen to circularize RNA template for its high efficiency and low cost. SuperScript IV reverse transcriptase was found to be able to catalyze the RCRT reaction on the circular RNA template, and the reaction efficiency was enhanced by adding the nicking enzyme, Nb.BbvCI to the RCRT system. E. Coli RNA polymerase, instead of the commonly used T7 RNA polymerase, was applied to synthesize long-strand RNA product for its high universality and processivity. Under the optimized conditions, small RNAs can be precisely amplified by 105∼6 folds. The fidelity of the established method was demonstrated by the accordance of the sequencing result and the initial RNA sequences. Free from expensive thermal cycler (necessary for RT-PCR-based amplification), precise replication of the initial RNA and high fidelity will enable the established strategy to be applied in RNA-seq, mRNA profiling, microarray analysis and RNA-based SELEX as well.

Selection of highly specific aptamers to Vibrio parahaemolyticus using cell-SELEX powered by functionalized graphene oxide and rolling circle amplification.

Cell-SELEX is a powerful tool to screen aptamers binding to living cellular organisms such as bacteria, fungi and even oncocytes. Here, we developed an advanced cell-SELEX strategy featuring functionalized graphene oxide (GO) and isothermal rolling circle amplification (RCA) to select aptamers against a prevailing foodborne pathogen, Vibrio parahaemolyticus. Polyethyleneglycol (PEG) and chitosan (CTS) were grafted onto the sheet-like GO molecules to synthesize a PC-GO material. TEM and FT-IR characterization demonstrated that the PC-GO composites were near-nanometric scale and tethered with PEG and CTS moieties, a property that significantly improved its solubility in biological buffer solutions used in cell-SELEX process. PC-GO could bind with ssDNAs with lower affinities to target cells, therefore the selection efficiency is greatly enhanced. The cell-binding aptamer candidates (CACs) were amplified by 107 fold using complementary ring mediated (CRM-RCA), a created amplification method that generate single-stranded products, which could be directly used in the next round selection. As fueled by PC-GO and CRM-RCA, four highly specific aptamers with lowest Kd value of 10.3 ±â€¯2.5 nM were obtained. Flow cytometry analysis showed that all the four aptamers exhibited more than 75% binding affinity to V. parahaemolyticus than to other foodborne bacteria (less than 18%). Simple procedure, high efficiency, and free from expensive thermal cycler (required by PCR amplification) will enable the established strategy to find its applications in aptamer selecting against fungi, stem and cancerous cells as well.