Our research is dedicated to the investigation of the structure-function relationships in RNA, which we pursue by developing novel cross-disciplinary approaches based on mass spectrometry (MS). The greatest biomedical discoveries have always been propelled by major technological advances. At the same time, the greatest technological advances have always been motivated by the need to solve the most challenging biomedical problems. With the overarching goal of supporting the development of new therapeutic strategies, our research focuses on viral systems responsible for infectious diseases and cancer. Current projects involve the investigation of non-protein coding regions of the genomes of SARS-CoV-2, HIV-1, hepatitis C, and Zika virus, which carry out essential regulatory activities in viral replication. Determining the structure/dynamics of these RNAs and their complexes with nucleic acids, proteins, and small molecule ligands can lead to the elucidation of their mechanism of action and the identification of new therapeutic targets. The analysis of RNA post-transcriptional modifications (rPTMs) can provide valuable insights into their regulatory functions and afford the basis for the rapid diagnosis of viral infection. Our projects combine biophysical, computational, bioinformatics, genomics, molecular biology, and virology approaches with new MS techniques to enable the acquisition of otherwise unattainable information and provide the unique insights necessary to move the field forward.

Sample purity, material availability, size limitations, and poor crystallization properties are just some of the factors that may prevent the direct application of classic structural approaches based on NMR and crystallography. We have been developing strategies based on chemical/biochemical probing and high-resolution mass spectrometric detection (i.e., MS3D) for the structural elucidation of samples that are not amenable to classic methods.

We have employed new crosslinking reagents to obtain valid spatial constraints necessary to support molecular modeling. We have explored structure-specific nucleases that are sensitive to the higher-order structure of RNA substrates. These technologies allowed us to obtain the first full-fledged 3D structures of RNA, which were solved by using experimental constraints obtained by MS-based techniques. We are currently investigating the structure of the frameshifting region of the genome of SARS-CoV-2, which we have found capable of folding into distinctive alternative forms.  Comparing such structures could provide the information necessary to design new inhibitors capable of preventing their interconversion.

The function of non-coding sequences of the human genome, which are estimated to encompass over 98% of the total 3-billion base pairs, is sustained by their ability to fold into well-defined structures capable of interacting with very specific cellular components. We are developing MS-based approaches for investigating the dynamics of long-range interactions that stabilize the higher-order structure of RNA and ribonucleoprotein. Tandem MS allowed us to investigate the process of dimerization and isomerization of the HIV-1 dimerization initiation site (DIS),which is mediated by the viral chaperone nucleocapsid protein (NC).

We are now utilizing ion mobility spectrometry (IMS) mass spectrometry to observe the effects of environmental conditions, metals, and other ligands on the stability of RNA structures. We designed a heated-emitter apparatus to observe the unfolding dynamics and determine the conformational stability of RNA. Unlike classic structural methods, IMS-MS enables the simultaneous detection of multiple conformations that are present in solution at the same time. This feature offers the ability to monitor the conformation of interconverting systems, such as riboswitches and ribozymes, which can assume alternative forms during their activity cycle.

The binding of proteins, metal ions, and a wide range of ligands can have profound effects on the activity of non-coding RNAs. Evaluating the stability of binding interactions and elucidating their structural determinants are crucial to understand their biological significance. We have developed MS-based approaches for the determination of dissociation constants of protein-RNA and ligand-RNA complexes, which were validated by using isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and fluorescence anisotropy techniques. We have explored the implementation of competitive binding experiments to rank the binding activity of close analogs that vied simultaneously for the same target, thus obtaining valuable information about their binding determinants.
We have developed approaches based on tandem MS, which enable the characterization of binding sites and interfaces between bound components. IMS-MS analyses have shown that the alternative conformers folded by RNA constructs in solution afford remarkably different affinities towards the various ligands, which implies the formation of distinct binding sites with unique characteristics. Additionally, this technique has been providing us with valuable new information on the mechanism of action of nucleic acid chaperones, such as the above-mentioned HIV-1 NC protein and the N-terminal RNA binding domain (NRBD) of the SARS-CoV-2 N-protein.

Natural RNA is decorated by over 170 different types of RNA post-transcriptional modifications (rPTMs), which have the ability to affect folding and intermolecular interactions. The fact that such modifications are established, removed, and interpreted by specific protein factors affords the opportunity to place RNA function under the control of signaling pathways involved in the response to stress, infection, and disease. We have developed a novel approach for the global analysis of all the rPTMs expressed by a certain cell at any given time, which allows the investigation of the epitranscriptomics regulation of gene expression. We are employing this approach to explore the possible relationships between essential processes of viral replication and the signaling pathways of different drugs of abuse, which are mediated by a subset of selected rPTMs. This information promises to shed new light onto the mechanism by which such drugs promote the re-emergence of HIV-1 from latency, which could lead to new therapeutic strategies. With collaborators, we are also capitalizing on this approach to understand how hepatitis C and Zika virus employ rPTMs to disguise their genomes and mRNAs as host RNA to elude the innate immune response and take over the normal metabolic machinery.

The type and abundance of rPTMs is determined by a combination of factors, including the genetic makeup of the organism under consideration, the possible exposure to environmental factors, and the general metabolic and physiologic state of the cell. This observation affords the opportunity to utilize rPTM  profiles obtained from total RNA extracts to evaluate the health of the cell and recognize diseases caused by possible RNA disfunction or viral/bacterial infection. We have developed an approach that combines global rPTM analysis with machine learning (ML) to generate classifiers capable of detecting pathogens in cell cultures and biological fluids. We are now testing the ability of this approach to correctly discriminate samples containing regular or drug-resistant strains. We have recently initiated a collaboration aimed at detecting the causative agents of sepsis in human synovial fluid, which could provide the means to achieve the rapid diagnosis of post-surgery infections necessary to significantly improve patient outcomes. This pilot project will provide proof-of-principle for a wide range of diagnostic applications that would substantiate the definitive transfer of our technology from lab bench to bedside.