Understanding hatchet ribozymes

Understanding hatchet ribozymes from chemical synthesis and crystallography to single-molecule fluorescence (FRET) and relaxation dispersion RNA NMR spectroscopy: Four novel self-cleaving ribozyme classes termed twister, twister-sister, pistol and hatchet have been discovered recently in the Breaker laboratory. Three of them (twister, twister-sister, and pistol ribozymes) have been investigated from distinct perspectives by several research groups (including us), resulting in a basic although still incomplete molecular understanding of how these ribozymes function. One of them, the hatchet ribozyme class has remained neglected and the secrets of its structure and mechanism have not been revealed yet. Hatchet ribozymes will be challenging in terms of structure elucidation and mode of action because of their apparent structural flexibility. The multidisciplinary approach designed for this project can tackle such a complex problem in RNA research: our undertaking is based on tailored, chemically modified RNAs and employs a broad set of methodologies ranging from classical to cutting-edge techniques. The goal of this project is to elucidate the principles that underlie catalysis of chemical reactions in conformationally flexible, highly dynamic RNA structures using hatchet ribozymes as model. We will accomplish this goal by harnessing the power of organic synthesis (i.e. synthesis of modified nucleosides; synthesis of site-specifically labeled RNA by solid-phase synthesis), biochemistry (i.e. atom-specific mutagenesis and nucleoside mutagenesis; Selective 2` Hydroxyl Acylation analyzed by Primer Extension (SHAPE) probing), and biophysics (X-ray crystallography; single-molecule Fluorescence-Resonance- Energy-Transfer(smFRET) imaging;relaxation dispersion CPMGandR1rhoNMR experiments). With this combined approach, we will be able to reach an unprecedented level of understanding of the structural dynamics-driven concepts that hatchet ribozymes apply to achieve reactivity. The structural flexibility of hatchet ribozymes is reminiscent of intrinsically disordered proteins (IDPs) that have been implicated in a number of diseases and that have been key to understand protein molecular recognition. Exploring the dynamics of RNA not only furthers RNA research in general but is essential to progress in biomedicine such as the advancement of small molecules (e.g., Ribocil, Branaplam) that bind therapeutic RNA targets. A detailed understanding of how structural dynamics determine RNA cleavage site recognition and RNA catalysis will certainly have crucial implications for future RNA-drug development.

Understanding ribozymes - From chemical synthesis and crystallography to fluorescence (FRET) and NMR spectroscopic analysis: Four new self-cleaving ribozyme classes, termed Twister, Twister-Sister, Pistol and Hatchet, were discovered in 2015 in the lab of Ronald Breaker (Yale). One of them (Twister) has been studied by different research groups (including us) from different perspectives, leading to a fundamental, albeit still incomplete, molecular understanding of how this ribozyme class works. Three of them, the hatchet, twister-sister, and pistol ribozymes, have been neglected so far and the secrets of their structure and mechanism had not yet been unraveled at the beginning of the project. These ribozymes pose a challenge for structural elucidation and mode of action due to their apparent structural flexibility. The multidisciplinary approach developed for this project can address such a complex problem in RNA research: Our project was based on customized, chemically modified RNAs and used a wide range of methods, ranging from classical to state-of-the-art techniques. The aim of the project was to elucidate the principles underlying the catalysis of chemical reactions in conformationally flexible, highly dynamic RNA structures, using hatchet and especially pistol ribozymes as models. To achieve this goal, organic synthesis (i.e. synthesis of modified nucleosides; synthesis of site-specifically labeled RNA by solid-phase synthesis), biochemistry (i.e. atom-specific mutagenesis and nucleoside mutagenesis; selective 2`-hydroxyl acylation analyzed by primer extension (SHAPE)) and biophysics (X-ray crystallography; fluorescence resonance energy transfer (FRET) spectroscopy; NMR experiments) was successfully applied. With this combined approach, we were able to gain an unprecedented level of understanding of the structural dynamics-driven concepts that ribozymes employ to achieve reactivity. The structural flexibility of ribozymes is reminiscent of intrinsically disordered proteins (IDPs), which play a role in a number of diseases and are crucial for understanding the molecular recognition of proteins. The study of RNA dynamics not only advances RNA research in general, but is also crucial for progress in biomedicine, for example for the development of small molecules (e.g. Ribocil, Branaplam) that bind therapeutic RNA targets. A detailed understanding of how structural dynamics determine RNA cleavage site recognition and RNA catalysis will certainly have crucial implications for future RNA drug development.