Toehold mediated strand displacement to measure released product from self-cleaving ribozymes

This paper presents a probe comprising a fluorophore and a quencher, enabling measurement of released product from self-cleaving hammerhead ribozyme, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labelled with a fluorophore at its 5′-end, while the other strand is labelled with a quencher at its 3′-end. These two DNA strands are perfectly complementary, but with a 3′-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the product release from ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

Rolling Circle RNA Synthesis Catalysed by RNA

RNA-catalysed RNA replication is widely considered a key step in the emergence of life's first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. RCS on small circular RNAs - as indicated by molecular dynamics simulations - induces a progressive build-up of conformational strain with destabilisation of nascent strand 5′ and 3′ ends. At the same time, we observe sustained RCS by a triplet polymerase ribozyme on small circular RNAs over multiple orbits with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

Kinetic Study of the Avocado Sunblotch Viroid Self-Cleavage Reaction Reveals Compensatory Effects between High-Pressure and High-Temperature: Implications for Origins of Life on Earth

A high pressure apparatus allowing one to study enzyme kinetics under pressure was used to study the self-cleavage activity of the avocado sunblotch viroid. The kinetics of this reaction were determined under pressure over a range up to 300 MPa (1–3000 bar). It appears that the initial rate of this reaction decreases when pressure increases, revealing a positive ΔV≠ of activation, which correlates with the domain closure accompanying the reaction and the decrease of the surface of the viroid exposed to the solvent. Although, as expected, temperature increases the rate of the reaction whose energy of activation was determined, it appeared that it does not significantly influence the ΔV≠ of activation and that pressure does not influence the energy of activation. These results provide information about the structural aspects or this self-cleavage reaction, which is involved in the process of maturation of this viroid. The behavior of ASBVd results from the involvement of the hammerhead ribozyme present at its catalytic domain, indeed a structural motif is very widespread in the ancient and current RNA world.

An ancient clade of Penelope-like retroelements with permuted domains is present in the green lineage and protists, and dominates many invertebrate genomes

ABSTRACTPenelope-like elements (PLEs) are an enigmatic clade of retroelements whose reverse transcriptases (RTs) share a most recent common ancestor with telomerase RTs. The single ORF of canonical EN+ PLEs encodes RT and a C-terminal GIY-YIG endonuclease (EN) that enables intrachromosomal integration, while EN–PLEs lack endonuclease and are generally restricted to chromosome termini. EN+ PLEs have only been found in animals, except for one case of horizontal transfer to conifers, while EN–PLEs occur in several kingdoms. Here we report a new, deep-branching PLE clade with a permuted domain order, whereby an N-terminal GIY-YIG endonuclease is linked to a C-terminal RT by a short domain with a characteristic Zn-finger-like motif. These N-terminal EN+ PLEs share a structural organization, including pseudo-LTRs and complex tandem/inverted insertions, with canonical EN+ PLEs from Penelope/Poseidon, Neptune and Nematis clades, and show insertion bias for microsatellites, but lack hammerhead ribozyme motifs. However, their phylogenetic distribution is much broader. The Naiad clade is found in numerous invertebrate phyla, where they can reach tens of thousands of copies per genome. Naiads in spiders and clams independently evolved to encode selenoproteins. Chlamys, which lack the CCHH motif universal to PLE endonucleases, occur in green algae, spike mosses (targeting ribosomal DNA) and the slime mold Physarum. Unlike canonical PLEs, RTs of N-terminal EN+ PLEs contain the insertion-in-fingers domain, strengthening the link between PLEs and telomerases. Additionally, we describe Hydra, a novel metazoan C-terminal EN+ clade. Overall, we conclude that PLE diversity, distribution and abundance is comparable to non-LTR and LTR-retrotransposons.

Hydrophobic-cationic peptides enhance RNA polymerase ribozyme activity by accretion

ABSTRACTAccretion and the resulting increase in local concentration to enhance target stability and function is a widespread mechanism in biology (for example in the liquid-liquid demixing phases and coacervates). It is widely believed that such macromolecular aggregates (formed through ionic and hydrophobic interactions) may have played a role in the origin of life. Here, we report on the behaviour of a hydrophobic-cationic RNA binding peptide selected by phage display (P43: AKKVWIIMGGS) that forms insoluble aggregates, accrete RNA on their surfaces in a size-dependent manner, and thus enhance the activities of various ribozymes. At low Mg2+ concentrations ([Mg2+]: 25 mM MgCl2), the activity of a small ribozyme (hammerhead ribozyme) was enhanced by P43, while larger ribozymes (RNA polymerase ribozyme (RPR), RNase P, F1* ligase) were inhibited. In contrast, at high [Mg2+] (≥200 mM), the RPR activity was enhanced. Another hydrophobic-cationic peptide with a simpler sequence (K2V6: KKVVVVVV) also exhibited similar regulatory effects on the RPR activity. Furthermore, inactive RPR captured on P43 aggregates at low [Mg2+] could be reactivated in a high [Mg2+] buffer. Therefore, in marked contrast to previously studied purely cationic peptides (like K10) that enhance RPR only at low ionic strength, hydrophobic-cationic peptides can reversibly concentrate RNA and enhance the RPR activity even at high ionic strength conditions such as in eutectic ice phases. Such peptides could have aided the emergence of longer and functional RNAs in a fluctuating environment (e.g., dry-wet / freeze-thaw cycles) on the prebiotic earth.

The model structure of the hammerhead ribozyme formed by RNAs of reciprocal chirality

RNA-based tools are frequently used to modulate gene expression in living cells. However, the stability and effectiveness of such RNA-based tools is limited by cellular nuclease activity. One way to increase RNA’s resistance to nucleases is to replace its D-ribose backbone with L-ribose isomers. This modification changes chirality of an entire RNA molecule to L-form giving it more chance of survival when introduced into cells. Recently, we have described the activity of left-handed hammerhead ribozyme (L-Rz, L-HH) that can specifically hydrolyze RNA with the opposite chirality at a predetermined location. To understand the structural background of the RNA specific cleavage in a heterochiral complex, we used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy as well as performed molecular modelling and dynamics simulations of homo- and heterochiral RNA complexes. The active ribozyme-target heterochiral complex showed a mixed chirality as well as low field imino proton NMR signals. We modelled the three dimensional structures of the oligoribonucleotides with their ribozyme counterparts of reciprocal chirality. L- or D-ribozyme formed a stable, homochiral helix 2, and two short double heterochiral helixes 1 and 3 of D- or L-RNA strand thorough irregular Watson-Crick base pairs. The formation of the heterochiral complexes is supported by the result of simulation molecular dynamics. These new observations suggest that L-catalytic nucleic acids can be used as tools in translational biology and diagnostics.

A synthetic RNA-based biosensor for fructose-1,6-bisphosphate that reports glycolytic flux

ABSTRACTMetabolic heterogeneity, the occurrence of different metabolic phenotypes among cells, represents a key challenge in health and biotechnology. To unravel its molecular basis, tools probing metabolism of single cells are needed. While RNA devices harbor huge potential for the development of such tools, until today, it is challenging to create in vivo-functional sensors for any given metabolite. Here, we developed from scratch an RNA-based sensor for fructose-1,6-bisphosphate (FBP), a doubly phosphorylated intermediate of glycolysis. Starting from in vitro selection of an RNA aptamer and its structural analyses, we developed libraries of RNA-based regulatory devices with this aptamer and the hammerhead ribozyme as an actuator. Through FACS-seq-based high-throughput screening in yeast, we identified in vivo-functional FBP-sensing devices that generate fluorescent readout dependent on intracellular FBP concentration. As FBP reports the flux through glycolysis, the developed RNA device can be used to sense the glycolytic rate in single cells, offering unprecedented possibilities to investigate the causes of metabolic heterogeneity.

Measurement of Kinetics of Hammerhead Ribozyme Cleavage Reactions using Toehold Mediated Strand Displacement

ABSTRACTThis paper presents a probe comprising a fluorophore and a quencher, enabling measurement of hammerhead ribozyme cleavage reactions, without labeled RNA molecules, regular sampling or use of polyacrylamide gels. The probe is made of two DNA strands; one strand is labelled with a fluorophore at its 5’-end, while the other strand is labelled with a quencher at its 3’-end. These two DNA strands are perfectly complementary, but with a 3’-overhang of the fluorophore strand. These unpaired nucleotides act as a toehold, which is utilized by a detached cleaved fragment (coming from a self-cleaving hammerhead ribozyme) as the starting point for a strand displacement reaction. This reaction causes the separation of the fluorophore strand from the quencher strand, culminating in fluorescence, detectable in a plate reader. Notably, the emitted fluorescence is proportional to the amount of detached cleaved-off RNAs, displacing the DNA quencher strand. This method can replace or complement radio-hazardous unstable 32P as a method of measurement of the kinetics of ribozyme cleavage reactions; it also eliminates the need for polyacrylamide gels, for the same purpose. Critically, this method allows to distinguish between the total amount of cleaved ribozymes and the amount of detached fragments, resulting from that cleavage reaction.

An antiviral self-replicating molecular heterotroph

We report the synthesis of a molecular machine, fabricated from nucleic acids, which is capable of digesting viral RNA and utilizing it to assemble additional copies of itself inside living cells. The machine’s body plan combines several parts that build upon the target RNA, assembling an immobile, DNA:RNA 4-way junction, which contains a single gene encoding a hammerhead ribozyme (HHR). Full assembly of the machine’s body from its parts enables the subsequent elongation of the gene and transcription of HHR molecules, followed by HHR-mediated digestion of the target molecule. This digestion converts the target to a building block suitable for participation in the assembly of more copies of the machine, mimicking biological heterotrophy. In this work we describe the general design of a prototypical machine, characterize its activity cycle and kinetics, and show that it can be efficiently and safely delivered into live cells. As a proof of principle, we constructed a machine that targets the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) GP64 gene, and show that it effectively suppresses viral propagation in a cell population, exhibiting predator/prey-like dynamics with the infecting virus. In addition, the machine significantly reduced viral infection, stress signaling, and innate immune activation inside virus-infected animals. This preliminary design could control the behavior of antisense therapies for a range of applications, particularly against dynamic targets such as viruses and cancer.