Search for Low-Mass Axion Dark Matter with ABRACADABRA-10 cm.

Two of the most pressing questions in physics are the microscopic nature of the dark matter that comprises 84% of the mass in the Universe and the absence of a neutron electric dipole moment. These questions would be resolved by the existence of a hypothetical particle known as the quantum chromodynamics (QCD) axion. In this work, we probe the hypothesis that axions constitute dark matter, using the ABRACADABRA-10 cm experiment in a broadband configuration, with world-leading sensitivity. We find no significant evidence for axions, and we present 95% upper limits on the axion-photon coupling down to the world-leading level g_{aγγ}<3.2×10^{-11} GeV^{-1}, representing one of the most sensitive searches for axions in the 0.41-8.27 neV mass range. Our work paves a direct path for future experiments capable of confirming or excluding the hypothesis that dark matter is a QCD axion in the mass range motivated by string theory and grand unified theories.

First Results from ABRACADABRA-10 cm: A Search for Sub-µeV Axion Dark Matter.

The axion is a promising dark matter candidate, which was originally proposed to solve the strong-CP problem in particle physics. To date, the available parameter space for axion and axionlike particle dark matter is relatively unexplored, particularly at masses m_{a}≲1 µeV. ABRACADABRA is a new experimental program to search for axion dark matter over a broad range of masses, 10^{-12}≲m_{a}≲10^{-6} eV. ABRACADABRA-10 cm is a small-scale prototype for a future detector that could be sensitive to the QCD axion. In this Letter, we present the first results from a 1 month search for axions with ABRACADABRA-10 cm. We find no evidence for axionlike cosmic dark matter and set 95% C.L. upper limits on the axion-photon coupling between g_{aγγ}<1.4×10^{-10} and g_{aγγ}<3.3×10^{-9} GeV^{-1} over the mass range 3.1×10^{-10}-8.3×10^{-9} eV. These results are competitive with the most stringent astrophysical constraints in this mass range.

Automated design of hammerhead ribozymes and validation by targeting the PABPN1 gene transcript.

We present a new publicly accessible web-service, RiboSoft, which implements a comprehensive hammerhead ribozyme design procedure. It accepts as input a target sequence (and some design parameters) then generates a set of ranked hammerhead ribozymes, which target the input sequence. This paper describes the implemented procedure, which takes into consideration multiple objectives leading to a multi-objective ranking of the computer-generated ribozymes. Many ribozymes were assayed and validated, including four ribozymes targeting the transcript of a disease-causing gene (a mutant version of PABPN1). These four ribozymes were successfully tested in vitro and in vivo, for their ability to cleave the targeted transcript. The wet-lab positive results of the test are presented here demonstrating the real-world potential of both hammerhead ribozymes and RiboSoft. RiboSoft is freely available at the website http://ribosoft.fungalgenomics.ca/ribosoft/.

Single-molecule observation of the induction of k-turn RNA structure on binding L7Ae protein.

The k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection of the kinked structure, or a more active process in which the protein manipulates the RNA structure. We have devised a single-molecule experiment whereby immobilized L7Ae protein binds Cy3-Cy5-labeled RNA from free solution. We find that all bound RNA is in the kinked geometry, with no evidence for transitions to an extended form at the millisecond timescale of the camera. Furthermore, real-time binding experiments provide no evidence for a more extended intermediate even at the earliest times, at a time resolution of 16 ms. The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form.

Structure of the three-way helical junction of the hepatitis C virus IRES element.

The hepatitis C virus internal ribosome entry site (IRES) element contains a three-way junction that is important in the overall RNA conformation, and for its role in the internal initiation of translation. The junction also illustrates some important conformational principles in the folding of three-way helical junctions. It is formally a 3HS(4) junction, with the possibility of two alternative stacking conformers. However, in principle, the junction can also undergo two steps of branch migration that would form 2HS(1)HS(3) and 2HS(2)HS(2) junctions. Comparative gel electrophoresis and ensemble fluorescence resonance energy transfer (FRET) studies show that the junction is induced to fold by the presence of Mg(2+) ions in low micromolar concentrations, and suggest that the structure adopted is based on coaxial stacking of the two helices that do not terminate in a hairpin loop (i.e., helix IIId). Single-molecule FRET studies confirm this conclusion, and indicate that there is no minor conformer present based on an alternative choice of helical stacking partners. Moreover, analysis of single-molecule FRET data at an 8-msec resolution failed to reveal evidence for structural transitions. It seems probable that this junction adopts a single conformation as a unique and stable fold.

A structural database for k-turn motifs in RNA.

The kink-turn (k-turn) is a common structural motif in RNA that introduces a tight kink into the helical axis. k-turns play an important architectural role in RNA structures and serve as binding sites for a number of proteins. We have created a database of known and postulated k-turn sequences and three-dimensional (3D) structures, available via the internet. This site provides (1) a database of sequence and structure, as a resource for the RNA community, and (2) a tool to enable the manipulation and comparison of 3D structures where known.

Orientation of cyanine fluorophores terminally attached to DNA via long, flexible tethers.

Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5'-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5'-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15 °C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by ∼60° for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.

Formation of an active site in trans by interaction of two complete Varkud Satellite ribozymes.

The complete VS ribozyme comprises seven helical segments, connected by three three-way RNA junctions. In the presence of Mg(2+) ions, cleavage occurs within the internal loop of helix I. This requires the participation of a guanine (G638) within the helix I loop, and a remote adenine (A756) within an internal loop of helix VI. Previous structural studies have suggested that helix I docks into the fold of the remaining part of the ribozyme, bringing A756 and G638 close to the scissile phosphate to allow the cleavage reaction to proceed. We show here that while either A756C or G638A individually exhibit very low cleavage activity, a mixture of the two variants leads to cleavage of the A756C RNA, but not the G638A RNA. The rate of cleavage depends on the concentration of the VS G638A RNA, as expected for a bimolecular interaction. This regaining of cleavage activity by complementation indicates that helix I of one VS RNA can interact with another VS RNA molecule to generate a functional active site in trans.

The complete VS ribozyme in solution studied by small-angle X-ray scattering.

We have used small-angle X-ray solution scattering to obtain ab initio shape reconstructions of the complete VS ribozyme. The ribozyme occupies an electron density envelope with an irregular shape, into which helical sections have been fitted. The ribozyme is built around a core comprising a near-coaxial stack of three helices, organized by two three-way helical junctions. An additional three-way junction formed by an auxiliary helix directs the substrate stem-loop, juxtaposing the cleavage site with an internal loop to create the active complex. This is consistent with the current view of the probable mechanism of trans-esterification in which adenine and guanine nucleobases contributed by the interacting loops combine in general acid-base catalysis.

A simple and affordable kinetic assay of nucleic acids with SYBR Gold gel staining.

Labeling substrates or products are paramount in determining enzymatic kinetic parameters. Several options are available; many laboratories use either radioactive or fluorescent labeling because of their high sensitivity. However, those methods have their own drawbacks such as half-life decay, expensive and hazardous. Here, we propose a novel, simple, economical and fast alternative to substrate labeling for studying the kinetics of nucleic acids: post-migration gel staining with SYBR Gold. Cleavage rates similar to the ones reported in the literature for the I-R3 DNA-cleaving DNA enzyme in the presence of zinc chloride are an indication of the quality of the new method. Moreover, the activity of the hammerhead ribozyme was also monitored by our method to illustrate its versatility. This labeling-free method has several advantages such as its ease of use as well as cost effective and versatility with both non-structured and structured RNAs or DNAs.

Single Mutation in Hammerhead Ribozyme Favors Cleavage Activity with Manganese over Magnesium.

Hammerhead ribozymes are one of the most studied classes of ribozymes so far, from both the structural and biochemical point of views. The activity of most hammerhead ribozymes is cation-dependent. Mg2+ is one of the most abundant divalent cations in the cell and therefore plays a major role in cleavage activity for most hammerhead ribozymes. Besides Mg2+, cleavage can also occur in the presence of other cations such as Mn2+. The catalytic core of hammerhead ribozymes is highly conserved, which could contribute to a preference of hammerhead ribozymes toward certain cations. Here, we show a naturally occurring variation in the catalytic core of hammerhead ribozymes, A6C, that can favor one metallic ion, Mn2+, over several other cations.

Formation of the P1.1 pseudoknot is critical for both the cleavage activity and substrate specificity of an antigenomic trans-acting hepatitis delta ribozyme.

Hepatitis delta virus RNAs possess self-cleavage activities that produce 2',3'-cyclic phosphate and 5'-hydroxyl termini (i.e. cis-acting delta ribozyme). Trans-acting delta ribozymes have been engineered by removing a junction from the cis version, thereby producing one molecule possessing the substrate sequence and the other the catalytic domain. According to the pseudoknot model, the secondary structure of the delta ribozyme includes a pseudoknot (i.e. P1.1 stem) formed by two base pairs from residues of the L3 loop and J1/4 junction. A collection of 48 P1.1 stem mutants was synthesized in order to provide an original characterization of both the importance and the structure of this pseudoknot in a trans-acting version of the ribozyme. Several structural differences were noted compared to the results reported for cis-acting ribozymes. For example, a combination of two stable Watson-Crick base pairs composing the essential P1.1 stem was demonstrated to be crucial for a significant level of activity, while the cis version required only one base pair. In addition, we present the first physical evidences revealing that the composition of the P1.1 stem affects the substrate specificity for ribozyme cleavage. Depending on the residues forming the J1/4 junction, non-productive ribozyme-substrate complexes can be observed. This phenomenon is proposed to be important for further development of a gene-inactivation system based on delta ribozyme.

RNA Fluorescence with Light-Up Aptamers.

Seeing is not only believing; it also includes understanding. Cellular imaging with GFP in live cells has been transformative in many research fields. Modulation of cellular regulation is tightly regulated and innovative imaging technologies contribute to further understand cellular signaling and physiology. New types of genetically encoded biosensors have been developed over the last decade. They are RNA aptamers that bind with their cognate fluorogen ligands and activate their fluorescence. The emergence and the evolution of these RNA aptamers as well as their conversion into a wide spectrum of applications are examined in a global way.

Ribozyme-based gene-inactivation systems require a fine comprehension of their substrate specificities; the case of delta ribozyme.

The ability of ribozymes (i.e. RNA enzymes) to specifically recognize and subsequently catalyze the cleavage of an RNA substrate makes them attractive for the development of therapeutic tools for the inactivation of both viral RNAs and mRNAs associated with various diseases. Several applicable ribozyme models have been tested both in vitro and in a cellular environment, and have shown significant promise. However, several hurdles remain to be surpassed before we generate a useful gene-inactivation system based on a ribozyme. Among the most important requirements for further progress are a better understanding of the features that contribute to defining the substrate specificity for cleavage by a ribozyme, and the identification of the potential cleavage sites in a given target RNA. The goal of this review is to illustrate the importance of both of these factors at the RNA level in the development of any type of ribozyme based gene-therapy. This is achieved by reviewing the recent progress in both the structure-function relationships and the development of a gene-inactivation system of a model ribozyme, specifically delta ribozyme.

Finding instances of riboswitches and ribozymes by homology search of structured RNA with Infernal.

In the genomics era, computational tools are essential to extract information from sequences and annotate them to allow easy access to genes. Fortunately, many of these tools are now part of standard pipelines. As a consequence, a cornucopia of genomic features is available in multiple databases. Nevertheless, as novel genomes are sequenced and new structured RNAs are discovered, homology searches and additional analyses need to be performed. In this chapter, we propose simple ways of finding instances of riboswitches and ribozymes in databases or in unannotated genomes, as well as ways of finding variants that deviate from the typical consensus.

Examination of the folding pathway of the antigenomic hepatitis delta virus ribozyme reveals key interactions of the L3 loop.

With the goal of gaining insight into the tertiary structure of the hepatitis delta virus ribozyme, cross-linking experiments using 4-thiouridine residues introduced in either the 5'-end portion of the substrate, or at seven strategic positions within the ribozyme, were performed. Mapping of the newly formed covalent bonds in cross-linked species obtained under various conditions, as well as using several mutated ribozymes, permitted monitoring of the formation of the ribozyme-substrate complex as the ribozyme proceeded along the folding pathway. In order to aid visualization of the tertiary structure transformation, an in silico animation of the "on" folding pathway was developed. In combination with those of the cleavage assays of structured substrates, these data shed light on the key contribution of the L3 loop in the formation of an active tertiary complex.

Nucleobase catalysis in the hairpin ribozyme.

RNA catalysis is important in the processing and translation of RNA molecules, yet the mechanisms of catalysis are still unclear in most cases. We have studied the role of nucleobase catalysis in the hairpin ribozyme, where the scissile phosphate is juxtaposed between guanine and adenine bases. We show that a modified ribozyme in which guanine 8 has been substituted by an imidazole base is active in both cleavage and ligation, with ligation rates 10-fold faster than cleavage. The rates of both reactions exhibit bell-shaped dependence on pH, with pK(a) values of 5.7 +/- 0.1 and 7.7 +/- 0.1 for cleavage and 6.1 +/- 0.3 and 6.9 +/- 0.3 for ligation. The data provide good evidence for general acid-base catalysis by the nucleobases.

Cross-linking experiments reveal the presence of novel structural features between a hepatitis delta virus ribozyme and its substrate.

The kinetic pathway of a trans-acting delta ribozyme includes an essential structural rearrangement involving the P1 stem, a stem that is formed between the substrate and the ribozyme. We performed cross-linking experiments to determine the substrate position within the catalytic center of an antigenomic, trans-acting, delta ribozyme. Substrates that included a 4-thiouridine either in position -1, +4, or +8 (i.e., adjacent to the cleavage site, or located either in the middle of or at the 3'-end of the P1 stem, respectively) were synthesized and shown to be efficiently cleaved. Examination of the cross-linking conditions, the use of various mutated ribozymes, as well as the probing and characterization of the resulting ribozyme-substrate complexes, revealed several new features of the molecular mechanism: (1) the close proximity of several bases between nucleotides of the substrate and ribozyme; (2) the active ribozyme-substrate complex folds in a manner that docks the middle of the P1 stem on the P3 stem, while concomitantly the scissile phosphate is in close proximity to the catalytic cytosine; and, (3) some complexes appear to be compatible with being active intermediates along the folding pathway, while others seem to correspond to misfolded structures. To provide a model representation of these data, a three-dimensional structure of the delta ribozyme was developed using several RNA bioinformatic software packages.