Deciphering ACE2-RBD binding affinity through peptide scanning: A molecular dynamics simulation approach.

Rapid discovery of target information for protein-protein interactions (PPIs) is significant in drug design, diagnostics, vaccine development, antibody therapy, etc. Peptide microarray is an ideal tool for revealing epitope information of PPIs. In this work, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) and the host cell receptor angiotensin-converting enzyme 2 (ACE2) were introduced as a model to study the epitope information of RBD-specific binding to ACE2 via a combination of theoretical calculations and experimental validation. Through dock and molecular dynamics simulations, it was found that among the 22 peptide fragments that consist of RBD, #14 (YNYLYRLFRKSNLKP) has the highest binding strength. Subsequently, the experiments of peptide microarray constructed based on plasmonic materials chip also confirmed the theoretical calculation data. Compared to other methods, such as phage display technology and surface plasmon resonance (SPR), this method is rapid and cost-effective, providing insights into the investigation of pathogen invasion processes and the timely development of peptide drugs and other fields.

Amplification-free microRNA profiling with femtomolar sensitivity on a plasmonic enhanced fluorescence nano-chip.

MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules involved in the regulation of gene expression, thus considered as promising biomarkers for cancer, cardiovascular diseases, neurodegenerative diseases, etc. However, quantitative analysis of miRNAs faces challenges owing to their high homology, small size & ultra-low abundance, and disease occurrence is often related to abnormal expression of multiple miRNAs where method for parallel miRNAs analysis is required. In this work, multiplexed analysis of miRNAs was established on a plasmonic nano-chip capable of fluorescence enhancement in the near-infrared region. Combined with polyadenylation at the hydroxyl terminate of target miRNA to afford abundant sites for fluorophore labeling, our assay achieved amplification-free detection of miRNAs from nM to fM with the limit of detection down to ca. 5 fM. A miRNA panel was constructed to detect 10 miRNAs differentially expressed in MCF-7 and A549 cell lines and validated with qRT-PCR, demonstrating the practical application of this method. This scalable platform can be customized for different miRNA panels, facilitating multiple miRNA profiling for various diseases.

A Novel Cell Membrane-Associated RNA Extraction Method and Its Application in the Discovery of Breast Cancer Markers.

Cell membrane-associated RNA (mem-RNA) has been demonstrated to be cell-specific and disease-related and are considered as potential biomarkers for disease diagnostics, drug delivery, and cell screening. However, there is still a lack of methods specifically designed to extract mem-RNA from cells, limiting the discovery and applications of mem-RNA. In this study, we propose the first all-in-one solution for high-purity mem-RNA isolation based on two types of magnetic nanoparticles, named MREMB (Membrane-associated RNA Extraction based on Magnetic Beads), which achieved ten times enrichment of cell membrane components and over 90% recovery rate of RNA extraction. To demonstrate MREMB's potential in clinical research, we extracted and sequenced mem-RNA of typical breast cancer MCF-7, MDA-MB-231, and SKBR-3 cell lines and non-neoplastic breast epithelial cell MCF-10A. Compared to total RNA, sequencing results revealed that membrane/secreted protein-encoding mRNAs and long noncoding RNAs (lncRNAs) were enriched in the mem-RNA, some of which were significantly overexpressed in the three cancer cell lines, including extracellular matrix-related genes COL5A1 and lncRNA TALAM1. The results indicated that MREMB could enrich membrane/secreted protein-coding RNA and amplify the expression differences of related RNAs between cancer and non-neoplastic cells, promising for cancer biomarker discovery.

A nano-magnetic size selective cfDNA extraction platform for liquid biopsy with enhanced precision.

As an emerging biomarker, cell-free DNA (cfDNA) carries crucial genetic information for the diagnosis of hereditary disease and cancer. However, test accuracy was severely compromised by the low abundance of cell-free DNA in peripheral blood, frequently diluted by genomic DNA released from white blood cells, resulting in sample rejection, test inaccuracy, and restricted clinical utility. Herein we report a novel strategy for the efficient recovery of cfDNA with significant removal of genomic DNA contamination during the cfDNA extraction process, based on a nano-magnetic size selective cfDNA extraction platform. With this platform, over 90% cfDNA recovery rate was achieved with minimal genomic DNA contamination. For non-invasive prenatal testing, an increase of fetal fraction from 10.10% to 29.94% medially was observed in 11 maternal plasma samples, with two false-negative samples identified by the proposed workflow. Enrichment of cfDNA in plasma sample of cancer patient demonstrated âˆ¼ 100% increase of circulating tumor DNA (ctDNA) percentage by panel sequencing of specific mutation sites. The approach is simple, automatable and cost-efficient, can improve liquid biopsy precision and reduce sequencing depth through significant enrichment of target abundance. The nano-magnetic platform demonstrated its potential application in liquid biopsy, since it exhibited numerous advantages in avoiding false negative results, reducing sequencing cost, improving data quality, and rescuing contaminated samples.

Biocatalytic Amplification of UV Signal in Capillary Electrophoresis of MicroRNA.

MicroRNAs (miRNAs) are new potential biomarkers for early diagnosis and classification of cancer. This study is the first attempt to use biocatalytic amplification reactions combined with capillary electrophoresis to detect multiple miRNAs simultaneously. In this way, miRNAs, as catalysts, can catalyze two single strands of DNA to form double-strand DNA. Feasibility was demonstrated by non-gel capillary electrophoresis coupled with UV detection (NGCE-UV). The detection limit was improved down to 1.0 nM, having ca. 103-fold improvement. This method has a good linear range of between 3.0 nM and 300 nM, with R2 at 0.99, recovery at 88-115%, and peak area precision at 1-12.7%. Using three target miRNAs as a model can achieve the baseline separation and good selectivity. The proposed biocatalysis coupled with a capillary electrophoresis-based method is simple, rapid, multiplexed, and cost-effective, making it potentially applicable for simultaneous, large-scale screening for other nucleic acids biomarkers and related research.