Structural basis for substrate binding and catalysis by a self-alkylating ribozyme.

Ribozymes that react with small-molecule probes have important applications in transcriptomics and chemical biology, such as RNA labeling and imaging. Understanding the structural basis for these RNA-modifying reactions will enable the development of better tools for studying RNA. Nevertheless, high-resolution structures and underlying catalytic mechanisms for members of this ribozyme class remain elusive. Here, we focus on a self-alkylating ribozyme that catalyzes nitrogen-carbon bond formation between a specific guanine and a 2,3-disubstituted epoxide substrate and report the crystal structures of a self-alkylating ribozyme, including both alkylated and apo forms, at 1.71-Å and 2.49-Å resolution, respectively. The ribozyme assumes an elongated hairpin-like architecture preorganized to accommodate the epoxide substrate in a hook-shaped conformation. Observed reactivity of substrate analogs together with an inverse, log-linear pH dependence of the reaction rate suggests a requirement for epoxide protonation, possibly assisted by the ether oxygens within the substrate.

Affinity maturation of a portable Fab-RNA module for chaperone-assisted RNA crystallography.

Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3-6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5'-GNGACCC-3' consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab-RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography.

A G-quadruplex-containing RNA activates fluorescence in a GFP-like fluorophore.

Spinach is an in vitro-selected RNA aptamer that binds a GFP-like ligand and activates its green fluorescence. Spinach is thus an RNA analog of GFP and has potentially widespread applications for in vivo labeling and imaging. We used antibody-assisted crystallography to determine the structures of Spinach both with and without bound fluorophore at 2.2-Å and 2.4-Å resolution, respectively. Spinach RNA has an elongated structure containing two helical domains separated by an internal bulge that folds into a G-quadruplex motif of unusual topology. The G-quadruplex motif and adjacent nucleotides comprise a partially preformed binding site for the fluorophore. The fluorophore binds in a planar conformation and makes extensive aromatic stacking and hydrogen bond interactions with the RNA. Our findings provide a foundation for structure-based engineering of new fluorophore-binding RNA aptamers.

Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR.

Site-directed spin labeling and pulsed electron-electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.

A Crystal Structure of a Functional RNA Molecule Containing an Artificial Nucleobase Pair.

As one of its goals, synthetic biology seeks to increase the number of building blocks in nucleic acids. While efforts towards this goal are well advanced for DNA, they have hardly begun for RNA. Herein, we present a crystal structure for an RNA riboswitch where a stem C:G pair has been replaced by a pair between two components of an artificially expanded genetic-information system (AEGIS), Z and P, (6-amino-5-nitro-2(1H)-pyridone and 2-amino-imidazo[1,2-a]-1,3,5-triazin-4-(8H)-one). The structure shows that the Z:P pair does not greatly change the conformation of the RNA molecule nor the details of its interaction with a hypoxanthine ligand. This was confirmed in solution by in-line probing, which also measured a 3.7 nM affinity of the riboswitch for guanine. These data show that the Z:P pair mimics the natural Watson-Crick geometry in RNA in the first example of a crystal structure of an RNA molecule that contains an orthogonal added nucleobase pair.

Nitroxide-labeled pyrimidines for non-covalent spin-labeling of abasic sites in DNA and RNA duplexes.

Non-covalent and site-directed spin labeling gives easy access to spin-labeled nucleic acids for the study of their structure and dynamics by electron paramagnetic resonance (EPR) spectroscopy. In a search for improved spin labels for non-covalent binding to abasic sites in duplex DNA and RNA, ten pyrimidine-derived spin labels were prepared in good yields and their binding was evaluated by continuous wave (CW)-EPR spectroscopy. Most of the spin labels showed lower binding affinity than the previously reported label ç towards abasic sites in DNA and RNA. The most promising labels were triazole-linked spin labels and a pyrrolocytosine label. In particular, the N1-ethylamino derivative of a triazole-linked uracil spin label binds fully to both DNA and RNA containing an abasic site. This is the first example of a spin label that binds fully through non-covalent interactions with an abasic site in RNA.

Structural changes of an abasic site in duplex DNA affect noncovalent binding of the spin label ç.

The influence of structural changes of an abasic site in duplex DNA on noncovalent and site-directed spin labeling (NC-SDSL) of the spin label ç were examined with electron paramagnetic resonance (EPR) spectroscopy. The binding affinities of ç to sixteen different DNA duplexes containing all possible sequences immediately flanking the abasic site were determined and the results showed that the binding of ç is highly flanking-sequence dependent. In general, a 5'-dG nucleotide favors the binding of the spin label. In particular, 5'-d(G__T) was the best binding sequence whereas 5'-d(C__T) showed the lowest affinity. Changing the structure of the abasic site linker from a tetrahydrofuran analog (F) to the anucleosidic C(3)-spacer (C(3)) does not appreciably affect the binding of ç to the abasic site. For efficient binding of ç, the abasic site needs to be located at least four base pairs away from the duplex end. Introducing a methyl substituent at N3 of ç did not change the binding affinity, but a decreased binding was observed for both N3-ethyl and -propyl groups. These results will guide the design of abasic site receptors and spin label ligands for NC-SDSL of nucleic acids.

Crystal structure of a DNA containing the planar, phenoxazine-derived bi-functional spectroscopic probe C.

Previously, we developed the deoxycytosine analog Ç (C-spin) as a bi-functional spectroscopic probe for the study of nucleic acid structure and dynamics using electron paramagnetic resonance (EPR) and fluorescence spectroscopy. To understand the effect of Ç on nucleic acid structure, we undertook a detailed crystallographic analysis. A 1.7 Å resolution crystal structure of Ç within a decamer duplex A-form DNA confirmed that Ç forms a non-perturbing base pair with deoxyguanosine, as designed. In the context of double-stranded DNA Ç adopted a planar conformation. In contrast, a crystal structure of the free spin-labeled base ç displayed a ∼ 20° bend at the oxazine linkage. Density function theory calculations revealed that the bent and planar conformations are close in energy and exhibit the same frequency for bending. These results indicate a small degree of flexibility around the oxazine linkage, which may be a consequence of the antiaromaticity of a 16-π electron ring system. Within DNA, the amplitude of the bending motion is restricted, presumably due to base-stacking interactions. This structural analysis shows that the Ç forms a planar, structurally non-perturbing base pair with G indicating it can be used with high confidence in EPR- or fluorescence-based structural and dynamics studies.

Effect of N3 modifications on the affinity of spin label ç for abasic sites in duplex DNA.

Noncovalent site-directed spin labeling (NC-SDSL) of abasic sites in duplex DNAs with the spin label ç, a cytosine analogue, is a promising approach for spin-labeling nucleic acids for EPR spectroscopy. In an attempt to increase the affinity of ç for abasic sites, several N3 derivatives were prepared, and their binding affinities were determined by EPR spectroscopy. Most of the N3 substituents had a detrimental effect on binding. The triazole-linked polyethylene-glycol derivative (12 a) showed a 15-fold decrease in affinity, whereas the binding affinities of ethyl azido (8 b) and hydroxyl (8 c) derivatives were five- to sixfold lower. The spin-labeled nucleoside Ç showed only a twofold decrease, thus binding better than 8 c, even though it contains the larger 2'-deoxyribose substituent at N3 instead of a 2-hydroxyethyl group. N3 derivatives that contained the basic ethyl amino (9) or ethyl guanidino (10) substituents had both higher binding affinity and solubility, attributed to their cationic charge at neutral pH. Compounds 9 and 10 are promising candidates for NC-SDSL of nucleic acids, for distance measurements by pulsed EPR spectroscopy.

Site-directed spin-labeling of nucleic acids by click chemistry: detection of abasic sites in duplex DNA by EPR spectroscopy.

This paper describes a spin label that can detect and identify local structural deformations in duplex DNA, in particular abasic sites. The spin label was incorporated into DNA by a new postsynthetic approach using click-chemistry on a solid support, which simplified both the synthesis and purification of the spin-labeled oligonucleotides. A nitroxide-functionalized azide, prepared by a short synthetic route, was reacted with an oligomer containing 5-ethynyl-2'-dU. The conjugation proceeded in quantitative yield and resulted in a fairly rigid linker between the modified nucleotide and the nitroxide spin label. The spin label was used to detect, for the first time, abasic sites in duplex DNA by X-band CW-EPR spectroscopy and give information about other structural deformations as well as local conformational changes in DNA. For example, reduced mobility of the spin label in a mismatched pair with T was consistent with the spin label displacing the T from the duplex. Addition of mercury(II) to this mispair resulted in a substantial increase in the motion of the spin label, consistent with formation of a metallopair between the T and the spin-labeled base that results in movement of the spin label out of the duplex and toward the solution. Thus, reposition of the spin label, when acting as a mercury(II)-controlled mechanical lever, can be readily detected by EPR spectroscopy. The ease of incorporation and properties of the new spin label make it attractive for EPR studies of nucleic acids and other macromolecules.

A conserved RNA structural motif for organizing topology within picornaviral internal ribosome entry sites.

Picornaviral IRES elements are essential for initiating the cap-independent viral translation. However, three-dimensional structures of these elements remain elusive. Here, we report a 2.84-Å resolution crystal structure of hepatitis A virus IRES domain V (dV) in complex with a synthetic antibody fragment-a crystallization chaperone. The RNA adopts a three-way junction structure, topologically organized by an adenine-rich stem-loop motif. Despite no obvious sequence homology, the dV architecture shows a striking similarity to a circularly permuted form of encephalomyocarditis virus J-K domain, suggesting a conserved strategy for organizing the domain architecture. Recurrence of the motif led us to use homology modeling tools to compute a 3-dimensional structure of the corresponding domain of foot-and-mouth disease virus, revealing an analogous domain organizing motif. The topological conservation observed among these IRESs and other viral domains implicates a structured three-way junction as an architectural scaffold to pre-organize helical domains for recruiting the translation initiation machinery.

Structural basis for activation of fluorogenic dyes by an RNA aptamer lacking a G-quadruplex motif.

The DIR2s RNA aptamer, a second-generation, in-vitro selected binder to dimethylindole red (DIR), activates the fluorescence of cyanine dyes, DIR and oxazole thiazole blue (OTB), allowing detection of two well-resolved emission colors. Using Fab BL3-6 and its cognate hairpin as a crystallization module, we solved the crystal structures of both the apo and OTB-SO3 bound forms of DIR2s at 2.0 Å and 1.8 Å resolution, respectively. DIR2s adopts a compact, tuning fork-like architecture comprised of a helix and two short stem-loops oriented in parallel to create the ligand binding site through tertiary interactions. The OTB-SO3 fluorophore binds in a planar conformation to a claw-like structure formed by a purine base-triple, which provides a stacking platform for OTB-SO3, and an unpaired nucleotide, which partially caps the binding site from the top. The absence of a G-quartet or base tetrad makes the DIR2s aptamer unique among fluorogenic RNAs with known 3D structure.

Spinach RNA aptamer detects lead(II) with high selectivity.

Spinach RNA aptamer contains a G-quadruplex motif that serves as a platform for binding and fluorescence activation of a GFP-like fluorophore. Here we show that Pb(2+) induces formation of Spinach's G-quadruplex and activates fluorescence with high selectivity and sensitivity. This device establishes the first example of an RNA-based sensor that provides a simple and inexpensive tool for Pb(2+) detection.