fluorescent-end-labeling-encapsulation-long-rnas-for-single-molecule

Fluorescent End-Labeling and Encapsulation of Long RNAs for Single-Molecule FRET-TIRF Microscopy

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) excels in studying dynamic biomolecules by allowing precise observation of their ...

University of Zurich

Fluorescence techniques for the investigation of biomolecules and their folding pathways require an efficient labeling strategy. A common method to internally label large RNAs involves the introduction of long loops for hybridization of fluorophore-carrying DNA strands. Such loops often disturb the structure, and thus the functionality, of the RNA. Here we show, in a proof of concept study with a >600 nucleotide group II intron ribozyme, that the usage of the nucleic acid analogue peptide nucleic acid (PNA) is more efficient in several aspects, minimizing the required structural modifications of the RNA. We demonstrate by various methods, including smFRET, that much smaller concentrations and shorter PNAs can be applied, compared to DNA, for rapid and specific internal RNA labeling. The folding pathway and catalytic activity of this large ribozyme is only minimally affected by the PNA, but the background signal is significantly reduced.

Strategy for Internal Labeling of Large RNAs with Minimal Perturbation by Using Fluorescent PNA.

Imaging fluorescently labeled biomolecules on a single-molecule level is a well-established technique to follow intra- and intermolecular processes in time, usually hidden in the ensemble average. The classical approach comprises surface immobilization of the molecule of interest, which increases the risk of restricting the natural behavior due to surface interactions. Encapsulation of such biomolecules into surface-tethered phospholipid vesicles enables to follow one molecule at a time, freely diffusing and without disturbing surface interactions. Further, the encapsulation allows to keep reaction partners (reactants and products) in close proximity and enables higher temperatures otherwise leading to desorption of the direct immobilized biomolecules.Here, we describe a detailed protocol for the encapsulation of a catalytically active RNA starting from surface passivation over RNA encapsulation to data evaluation of single-molecule FRET experiments in TIRF microscopy. We present an optimized procedure that preserves RNA functionality and applies to investigations of, e.g., large ribozymes and RNAs, where direct immobilization is structurally not possible.

Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy.

We coupled SPR imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) to identify new potential RNA binders. Here, we improve this powerful method, especially by optimizing the proteolytic digestion (type of reducing agent, its concentration, and incubation time), to work with complex mixtures, specifically a lysate of the rough mitochondrial fraction from yeast. The advantages of this hyphenated method compared to column-based or separate analyses are (i) rapid and direct visual readout from the SPRi array, (ii) possibility of high-throughput analysis of different interactions in parallel, (iii) high sensitivity, and (iv) no sample loss or contamination due to elution or micro-recovery procedures. The model system used is a catalytically active RNA (group IIB intron from Saccharomyces cerevisiae, Sc.ai5γ) and its cofactor Mss116. The protein supports the RNA folding process and thereby the subsequent excision of the intronic RNA from the coding part. Using the novel approach of coupling SPR with MALDI MS, we report the identification of potential RNA-binding proteins from a crude yeast mitochondrial lysate in a non-targeted approach. Our results show that proteins other than the well-known cofactor Mss116 interact with Sc.ai5γ (Dbp8, Prp8, Mrp13, and Cullin-3), suggesting that the intron folding and splicing are regulated by more than one cofactor in vivo.

Screening for potential interaction partners with surface plasmon resonance imaging coupled to MALDI mass spectrometry.

Fast and efficient site-specific labeling of long RNAs is one of the main bottlenecks limiting distance measurements by means of Förster resonance energy transfer (FRET) or electron paramagnetic resonance (EPR) spectroscopy. Here, we present an optimized protocol for dual end-labeling with different fluorophores at the same time meeting the restrictions of highly labile and degradation-sensitive RNAs. We describe in detail the dual-labeling of a catalytically active wild-type group II intron as a typical representative of long functional RNAs. The modular procedure chemically activates the 5'-phosphate and the 3'-ribose for bioconjugation with a pair of fluorophores, as shown herein, or with spin labels. The mild reaction conditions preserve the structural and functional integrity of the biomacromolecule and results in covalent, dual-labeled RNA in its pre-catalytic state in yields suitable for both ensemble and single-molecule FRET experiments.

Chemical Dual End-Labeling of Large Ribozymes.

RNA splicing, the removal of introns and ligation of exons, is a crucial process during mRNA maturation. Group II introns are large ribozymes that self-catalyze their splicing, as well as their transposition. They are living fossils of spliceosomal introns and eukaryotic retroelements. The yeast mitochondrial Sc.ai5γ is the first identified and best-studied self-splicing group II intron. A combination of biochemical, biophysical, and computational tools enables studying its catalytic properties, structure, and dynamics, while also serving to develop new therapeutic and biotechnological tools. We survey the history of group II intron studies paralleling the trends in RNA methodology with Sc.ai5γ in the spotlight.

Group II Introns: Highly Structured yet Dynamic.

Trillions of microorganisms, collectively known as the microbiome, inhabit our bodies with the gut microbiome being of particular interest in biomedical research. Bacteriophages, the dominant virome constituents, can utilize suppressor tRNAs to switch to alternative genetic codes (e.g., the UAG stop-codon is reassigned to glutamine) while infecting hosts with the standard bacterial code. However, what triggers this switch and how the bacteriophage manipulates its host is poorly understood. Here, we report the discovery of a subgroup of minimal hepatitis delta virus (HDV)-like ribozymes - theta ribozymes - potentially involved in the code switch leading to the expression of recoded lysis and structural phage genes. We demonstrate their HDV-like self-scission behavior in vitro and find them in an unreported context often located with their cleavage site adjacent to tRNAs, indicating a role in viral tRNA maturation and/or regulation. Every fifth associated tRNA is a suppressor tRNA, further strengthening our hypothesis. The vast abundance of tRNA-associated theta ribozymes - we provide 1753 unique examples - highlights the importance of small ribozymes as an alternative to large enzymes that usually process tRNA 3'-ends. Our discovery expands the short list of biological functions of small HDV-like ribozymes and introduces a previously unknown player likely involved in the code switch of certain recoded gut bacteriophages.

Identification of HDV-like theta ribozymes involved in tRNA-based recoding of gut bacteriophages.

RNA, widely recognized as an information-carrier molecule, is capable of catalyzing essential biological processes through ribozymes. Despite their ubiquity, specific functions in a biological context and phenotypes based on the ribozymes' activity are often unknown. Here, we present the discovery of a subgroup of minimal HDV-like ribozymes, which reside 3' to viral tRNAs and appear to cleave the 3'-trailers of viral premature tRNA transcripts. This proposed tRNA-processing function is unprecedented for any ribozymes, thus, we designate this subgroup as theta ribozymes. Most theta ribozymes were identified in Caudoviricetes bacteriophages, the main constituent (>90%) of the mammalian gut virome. Intriguingly, our findings further suggest the involvement of theta ribozymes in the transition of certain bacteriophages between distinct genetic codes, thus possibly contributing to the phage lysis trigger. Our discovery expands the limited repertoire of biological functions attributed to HDV-like ribozymes and provides insights into the fascinating world of RNA catalysis.

Article	Year
Strategy for Internal Labeling of Large RNAs with Minimal Perturbation by Using Fluorescent PNA. Chembiochem : a European journal of chemical biology\| PubMed ID: 25872497	2015
Encapsulation of Fluorescently Labeled RNAs into Surface-Tethered Vesicles for Single-Molecule FRET Studies in TIRF Microscopy. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32006303	2020
Screening for potential interaction partners with surface plasmon resonance imaging coupled to MALDI mass spectrometry. Analytical biochemistry\| PubMed ID: 33857502	2021
Chemical Dual End-Labeling of Large Ribozymes. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35226323	2022
Group II Introns: Highly Structured yet Dynamic. Chimia\| PubMed ID: 38047803	2023
Identification of HDV-like theta ribozymes involved in tRNA-based recoding of gut bacteriophages. Nature communications\| PubMed ID: 38378708	2024
From Enigma to Revelation: Unravelling Biological Functions of Ubiquitous Small Ribozymes. Chimia\| PubMed ID: 38676609	2024

Susann Zelger-Paulus

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