Preliminary study on the effect of nucleolin specific aptamer�miRNA let-7d chimera on Janus kinase-2 expression level and activity in gastric cancer (MKN-45) cells

Recently, much attention has been focused on the use of miRNAs in cancer treatment. The role of proto-oncogene Janus kinase-2 (JAK-2) in proliferation and survival of gastric cancer has been previously documented. The aim of this study was to evaluate the effect of a chimera consisted of nucleolin specific aptamer (NCL-Apt) and miRNA let-7d on JAK2 expression level and activity in gastric cancer cells. NCL-Apt�miRNA let-7d chimera was prepared by two methods. Gastric cancer (MKN-45) cell line and control cell line of human dermal fibroblast (HDF) were treated with the chimera and the changes in JAK2 expression and activity were determined using real-time PCR and ELISA techniques, respectively. In MKN-45 cells, the chimera caused significant decrease in JAK2 expression level and activity compared to the aptamer alone and miRNA mimic negative control. Nevertheless, transfected miRNA let-7d showed remarkable reduction in the expression level of JAK2 in comparison with control state in both MKN-45 and HDF, confirmed unspecific effect of let-7d on normal and cancerous cells. With regard to the synergic effect of this chimera on JAK2 activity, it might be viewed as a therapeutic candidate in gastric cancer. However, further studies are warranted to prove it. © 2018, Springer Nature B.V

High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency

The systemic evolution of ligands by exponential enrichment (SELEX) technique is a powerful and effective aptamer-selection procedure. However, modifications to the process can dramatically improve selection efficiency and aptamer performance. For example, droplet digital PCR (ddPCR) has been recently incorporated into SELEX selection protocols to putatively reduce the propagation of byproducts and avoid selection bias that result from differences in PCR efficiency of sequences within the random library. However, a detailed, parallel comparison of the efficacy of conventional solution PCR versus the ddPCR modification in the RNA aptamer-selection process is needed to understand effects on overall SELEX performance. In the present study, we took advantage of powerful high throughput sequencing technology and bioinformatics analysis coupled with SELEX (HT-SELEX) to thoroughly investigate the effects of initial library and PCR methods in the RNA aptamer identification. Our analysis revealed that distinct “biased sequences” and nucleotide composition existed in the initial, unselected libraries purchased from two different manufacturers and that the fate of the “biased sequences” was target-dependent during selection. Our comparison of solution PCR- and ddPCR-driven HT-SELEX demonstrated that PCR method affected not only the nucleotide composition of the enriched sequences, but also the overall SELEX efficiency and aptamer efficacy

Prospects in the use of aptamers for characterizing the structure and stability of bioactive proteins and peptides in food

Food-derived bioactive proteins and peptides have gained acceptance among researchers, food manufacturers and consumers as health-enhancing functional food components that also serve as natural alternatives for disease prevention and/or management. Bioactivity in food proteins and peptides is determined by their conformations and binding characteristics, which in turn depend on their primary and secondary structures. To maintain their bioactivities, the molecular integrity of bioactive peptides must remain intact, and this warrants the study of peptide form and structure, ideally with robust, highly specific and sensitive techniques. Short single-stranded nucleic acids (i.e. aptamers) are known to have high affinity for cognate targets such as proteins and peptides. Aptamers can be produced cost-effectively and chemically derivatized to increase their stability and shelf life. Their improved binding characteristics and minimal modification of the target molecular signature suggests their suitability for real-time detection of conformational changes in both proteins and peptides. This review discusses the developmental progress of systematic evolution of ligands by exponential enrichment (SELEX), an iterative technology for generating cost-effective aptamers with low dissociation constants (K d ) for monitoring the form and structure of bioactive proteins and peptides. The review also presents case studies of this technique in monitoring the structural stability of bioactive peptide formulations to encourage applications in functional foods. The challenges and potential of aptamers in this research field are also discussed