ABSTRACT
Deep-UV resonance Raman spectroscopy has been shown to offer great potential for probing the in situ stability of mRNA vaccines. In this study, a vaccine model was subjected to controlled degradation using RNase A or through aging at room temperature. The degradation of mRNA was confirmed by using a cell transfection test and by gel electrophoresis. Under both settings, DUVRR spectroscopy successfully revealed the mRNA degradation signs of the vaccine model.
Subject(s)
Spectrum Analysis, Raman , mRNA Vaccines , Spectrum Analysis, Raman/methods , Protein Structure, SecondaryABSTRACT
The ever-growing demand for new drugs highlights the need to develop novel cost- and time-effective techniques for drug discovery. Surface-enhanced Raman spectroscopy (SERS) is an emerging ultrasensitive and label-free technique that allows for the efficient detection and characterization of molecular interactions. We have recently developed a SERS platform for detecting a single protein molecule linked to a gold substrate (Almehmadi et al. Scientific Reports 2019). In this study, we extended the approach to probe the binding of potential drugs to RNA targets. To demonstrate the proof of concept, two 16-amino acid residue peptides with close primary structures and different binding affinities to the RNA CUG repeat related to myotonic dystrophy were tested. Three-microliter solutions of the RNA repeat with these peptides at nanomolar concentrations were probed using the developed approach, and the binding of only one peptide was demonstrated. The SER spectra exhibited significant fluctuations along with a sudden strong enhancement as spectra were collected consecutively from individual spots. Principal component analysis (PCA) of the SER spectral datasets indicated that free RNA repeats could be differentiated from those complexed with a peptide with 100% accuracy. The developed SERS platform provides a novel opportunity for label-free screening of RNA-binding peptides for drug discovery. Schematic representation of the SERS platform for drug discovery developed in this study.
Subject(s)
Metal Nanoparticles , Spectrum Analysis, Raman , Drug Discovery , Metal Nanoparticles/chemistry , Peptides , RNA , Spectrum Analysis, Raman/methodsABSTRACT
A two-step process of protein detection at a single molecule level using SERS was developed as a proof-of-concept platform for medical diagnostics. First, a protein molecule was bound to a linker in the bulk solution and then this adduct was chemically reacted with the SERS substrate. Traut's Reagent (TR) was used to thiolate Bovine serum albumin (BSA) in solution followed by chemical cross linking to a gold surface through a sulfhydryl group. A Glycine-TR adduct was used as a control sample to identify the protein contribution to the SER spectra. Gold SERS substrates were manufactured by electrochemical deposition. Solutions at an ultralow concentration were used for attaching the TR adducts to the SERS substrate. Samples showed the typical behavior of a single molecule SERS including spectral fluctuations, blinking and Raman signal being generated from only selected points on the substrate. The fluctuating SER spectra were examined using Principle Component Analysis. This unsupervised statistics allowed for the selecting of spectral contribution from protein moiety indicating that the method was capable of detecting a single protein molecule. Thus we have demonstrated, that the developed two-step methodology has the potential as a new platform for medical diagnostics.
