The lab is developing novel RNAomics technologies for obtaining global profiles of all the RNA modifications expressed by a certain cell at any given time. There are over one hundred known variants of the canonical ribonucleotides. With a handful of exceptions, their biological function is largely unknown due to the lack of adequate approaches for detecting them, identifying their sequence position, and quantifying their expression. For this reason, we have developed strategies based on Ion Mobility Spectrometry mass spectrometry (IMS-MS), which enable the comprehensive analysis of modified ribonucleotides at the full-transcriptome level (Figure 1). Subtracting heat-maps obtained from diseased versus healthy cells can immediately reveal differences in the composition and content of modifications, which can be correlated to the metabolic state of the cells under investigation. We are employing these strategies as diagnostic and investigative tools to obtain new insights into cellular processes involved in cancer and infection diseases.
HIGHER ORDER STRUCTURE OF NUCLEIC ACIDS
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 (Figure 2). We are currently investigating the implementation of these strategies in vivo, with the goal of obtaining direct structure information from target samples immersed in their natural environment, in the presence of all cellular factors involved in their biological functions. Our favorite targets include selected regions of the genomes of human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), and polio virus.
NUCLEIC ACID DYNAMICS
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 ribonucleoproteins. Tandem mass spectrometry 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 IMS-MS to observe the effects of environmental conditions, metals, and other ligands on the stability of RNA structures (Figure 3). Unlike classic structural methods, this technique enables the simultaneous detection of multiple conformations that may be present in solution at the same time. This feature offers the ability to monitor the dynamics of systems that, like riboswitches and ribozymes, manifest significant conformational changes during activity.
A firm knowledge of the cellular components that establish mutual contacts with the RNA of interest is crucial to understand its mechanism of action and biological significance. In the case of non-coding RNAs, the specific binding of cognate factors may shape the structure of dynamic elements, thus enacting regulation of their activity. Conversely, the alternative conformers folded by such elements may recruit different types of factors that mediate different biological functions. For these reasons, we are exploring possible in vivo applications of bifunctional crosslinkers and similar reagents to identify any protein that may come into direct contact with selected RNA targets. In particular, we are investigating the interaction of host and viral proteins with the 5’-untranslated regions (5’-UTRs) of the genomes of HIV-1, HCV and polio. We plan on utilizing mutagenesis and knockdown approaches to elucidate the effects of RNA structure on the recruiting preferences of these targets. The results will provide new insights into the mechanism by which 5’-UTR coordinates many vital steps of viral replication.
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 data obtained from isothermal titration calorimetry (ITC) and electron plasmon resonance (EPR). 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 (Figure 4). We have developed approaches based on tandem mass spectrometry, which enable the characterization of binding sites and interfaces between bound components. We are now utilizing IMS-MS to monitor the dynamics of free versus bound RNA, which will provide valuable information on the conformational effects of ligand interactions. These technologies afford an enormous potential in drug discovery and development, which we are exploring by using selected targets from HIV-1, HCV and polio.
Elucidating the structure and dynamics of a certain system constitutes the first step toward the rational design of possible strategies aimed at modulating or inhibiting its functions. MS-based technologies offer the opportunity to perform the direct evaluation of the inhibitory properties of putative drug candidates onto selected interactions. In this direction, we have demonstrated the ability to determine the ligand concentration capable of inducing 50% inhibition (a.k.a. IC50) of selected protein-RNA complexes (Figure 5). We are utilizing MS analysis to evaluate the effects of drug candidates on the integrity of the zinc-finger domains of HIV-1 nucleocapsid protein (NC) and to observe the aftermath on its chaperone activities. We are also employing IMS-MS to investigate the effects of antisense oligonucleotides on the structure and dynamics of HIV-1 5’-UTR. The results promise to provide valuable insights not only for possible applications in HIV therapy, but also for guiding the design of improved antisense.
12T solariX FT-ICR Mass Spectrometer
Velos LTQ Orbitrap Mass Spectrometer
Synapt G2 HDMS Ion Mobility Mass Spectrometer