ABSTRACT
Charge transport in biomolecules is crucial for many biological and technological applications, including biomolecular electronics devices and biosensors. RNA has become the focus of research because of its importance in biomedicine, but its charge transport properties are not well understood. Here, we use the Scanning Tunneling Microscopy-assisted molecular break junction method to measure the electrical conductance of particular 5-base and 10-base single-stranded (ss) RNA sequences capable of base stacking. These ssRNA sequences show single-molecule conductance values around [Formula: see text] ([Formula: see text]), while equivalent-length ssDNAs result in featureless conductance histograms. Circular dichroism (CD) spectra and MD simulations reveal the existence of extended ssRNA conformations versus folded ssDNA conformations, consistent with their different electrical behaviors. Computational molecular modeling and Machine Learning-assisted interpretation of CD data helped us to disentangle the structural and electronic factors underlying CT, thus explaining the observed electrical behavior differences. RNA with a measurable conductance corresponds to sequences with overall extended base-stacking stabilized conformations characterized by lower HOMO energy levels delocalized over a base-stacking mediating CT pathway. In contrast, DNA and a control RNA sequence without significant base-stacking tend to form closed structures and thus are incapable of efficient CT.
Subject(s)
DNA , RNA , RNA/metabolism , DNA/chemistry , DNA, Single-Stranded , Molecular Conformation , Models, MolecularABSTRACT
The COVID-19 pandemic shows a critical need for rapid, inexpensive, and ultrasensitive early detection methods based on biomarker analysis to reduce mortality rates by containing the spread of epidemics. This can be achieved through the electrical detection of nucleic acids at the single-molecule level. In particular, the scanning tunneling microscopic-assisted break junction (STM-BJ) method can be utilized to detect individual nucleic acid molecules with high specificity and sensitivity in liquid samples. Here, we demonstrate single-molecule electrical detection of RNA coronavirus biomarkers, including those of SARS-CoV-2 as well as those of different variants and subvariants. Our target sequences include a conserved sequence in the human coronavirus family, a conserved target specific for the SARS-CoV-2 family, and specific targets at the variant and subvariant levels. Our results demonstrate that it is possible to distinguish between different variants of the COVID-19 virus using electrical conductance signals, as recently suggested by theoretical approaches. Our results pave the way for future miniaturized single-molecule electrical biosensors that could be game changers for infectious diseases and other public health applications.
Subject(s)
Biosensing Techniques , COVID-19 , Nucleic Acids , Humans , COVID-19/diagnosis , RNA , SARS-CoV-2 , PandemicsABSTRACT
Cancer is a significant healthcare issue, and early screening methods based on biomarker analysis in liquid biopsies are promising avenues to reduce mortality rates. Electrical detection of nucleic acids at the single molecule level could enable these applications. We examine the electrical detection of RNA cancer biomarkers (KRAS mutants G12C and G12V) as a single-molecule proof-of-concept electrical biosensor for cancer screening applications. We show that the electrical conductance is highly sensitive to the sequence, allowing discrimination of the mutants from a wild-type KRAS sequence differing in just one base. In addition to this high specificity, our results also show that these biosensors are sensitive down to an individual molecule with a high signal-to-noise ratio. These results pave the way for future miniaturized single-molecule electrical biosensors that could be groundbreaking for cancer screening and other applications.
Subject(s)
Biosensing Techniques , Neoplasms , Nucleic Acids , Humans , RNA , Biomarkers, Tumor/genetics , Biomarkers, Tumor/analysis , Proto-Oncogene Proteins p21(ras)/genetics , Neoplasms/diagnosis , Neoplasms/genetics , Biosensing Techniques/methods , BiomarkersABSTRACT
RNA oligonucleotides are crucial for a range of biological functions and in many biotechnological applications. Herein, we measured, for the first time, the conductance of individual double-stranded (ds)RNA molecules and compared it with the conductance of single DNA : RNA hybrids. The average conductance values are similar for both biomolecules, but the distribution of conductance values shows an order of magnitude higher variability for dsRNA, indicating higher molecular flexibility of dsRNA. Microsecond Molecular Dynamics simulations explain this difference and provide structural insights into the higher stability of DNA : RNA duplex with atomic level of detail. The rotations of 2'-OH groups of the ribose rings and the bases in RNA strands destabilize the duplex structure by weakening base stacking interactions, affecting charge transport, and making single-molecule conductance of dsRNA more variable (dynamic disorder). The results demonstrate that a powerful combination of state-of-the-art biomolecular electronics techniques and computational approaches can provide valuable insights into biomolecules' biophysics with unprecedented spatial resolution.
Subject(s)
Oligonucleotides , RNA, Double-Stranded , DNA/chemistry , Molecular Dynamics Simulation , Nucleic Acid Conformation , Oligonucleotides/chemistry , RNA/chemistryABSTRACT
The last half-century has witnessed the birth and development of a new multidisciplinary field at the edge between materials science, nanoscience, engineering, and chemistry known as Molecular Electronics. This field deals with the electronic properties of individual molecules and their integration as active components in electronic circuits and has also been applied to biomolecules, leading to BioMolecular Electronics and opening new perspectives for single-molecule biophysics and biomedicine. Herein, we provide a brief introduction and overview of the BioMolecular electronics field, focusing on nucleic acids and potential applications for these measurements. In particular, we review the recent demonstration of the first single-molecule electrical detection of a biologically-relevant nucleic acid. We also show how this could be used to study biomolecular interactions and applications in liquid biopsy for early cancer detection, among others. Finally, we discuss future perspectives and challenges in the applications of this fascinating research field.
