Sandip A. Shelke

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 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 pre-formed 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.

Noncovalent and site-directed spin labeling of nucleic acids.

Electron paramagnetic resonance (EPR) spectroscopy is widely used to study free radicals or paramagnetic centers associated with biopolymers. With the advent of pulsed EPR methods, which allow accurate distance measurements between 20 and 80 , structures of biopolymers have increasingly been interrogated by this technique. Some of the advantages of EPR spectroscopy over other structural techniques are its sensitivity, that it is not restricted by molecular size, and that measurements can be performed under biological conditions. However, stable free radicals, such as nitroxide spin labels, must be incorporated into the biopolymers prior to EPR studies. In site-directed spin labeling (SDSL), spin labels are covalently attached to the biopolymers at a specific site of interest. For nucleic acids there have been two main strategies for SDSL. First, spin labels have been incorporated during automated oligonucleotide synthesis by employing spin-labeled phosphoramidite building blocks. This approach has the advantage that very sophisticated and structurally complex labels can be incorporated at specific sites. However, the synthetic challenges of spin-labeled phosphoramidites can be considerable. Furthermore, spin labels can be partially reduced upon exposure to the reagents used in the automated synthesis of oligonucleotides. The second SDSL approach is post-synthetic modification of the biopolymer. Here, a spin-labeling reagent is incubated with an oligonucleotide that contains a reactive functional group at a specific site. Post-synthetic labeling is in general less labor intensive than the phosphoramidite strategy, but drawbacks include incomplete labeling and side reactions of the spin label with inherent functional groups of the nucleic acids, such as the exocyclic amino groups of the nucleobases. Both strategies usually require purification of the spin-labeled material, which can be nontrivial. Here we report a new and straightforward SDSL protocol for nucleic acids that is based on noncovalent labeling. The new approach utilizes a nitroxide that is structurally related to the rigid spin label Ç. The spin label Ç is an analogue of cytidine (C), with a nitroxide-bearing isoindol moiety fused to cytosine by an oxazine linkage, and forms a stable Watson–Crick base pair with guanine (Figure 1). The rigidity of Ç enabled precise distance measurements by EPR, determination of the relative angular orientation between two spin labels, and has been used to study DNA dynamics and folding. 7] The strategy for noncovalent labeling was to disconnect the glycosidic bond of Ç to give an abasic site (F) and the free spin-labeled base (Figure 1). The spin label would bind in the abasic site through receptor–ligand interactions involving hydrogen bonding and p-stacking interactions. The synthesis of spin label started with regioselective alkylation of 5-bromouracil at N1 by a one-pot, two-step procedure using HMDS and benzyl bromide in the presence of a catalytic amount of iodine to obtain compound 2 (Scheme 1). Activation of 2 by conversion to the Osulfonylated derivative, followed by coupling with isoindol amino phenol derivative 4 yielded conjugate 5. Subsequent ring closure, facilitated by cesium fluoride, yielded phenoxazine derivative 6. Removal of the N1-protecting benzyl group by boron tribromide and oxidation of the amine to a nitroxide with mCPBA gave spin label . The EPR spectrum of in an aqueous solution containing ethylene glycol (30%) shows three narrow lines that broaden on reducing the temperature from 0 to 30 8C (Figure 2, left), due to slower tumbling of in solution. On mixing a DNA duplex containing an abasic site with , a slow-moving component appears in the EPR spectrum (shown by arrows, Figure 2 middle), indicating binding of the spin label to the abasic site. On further cooling, the extent of spin-label binding increased, and at 30 8C the narrow lines (the fastmotion component of the spectrum) had completely disappeared, consistent with the spin label being fully bound. For comparison, EPR spectra of a covalently Ç-labeled 14-mer were recorded under identical conditions (Figure 2, right). The mobility of the spin label that is covalently linked to the dsDNA is the same as that of the slow-moving component in the sample containing and the abasic DNA (Figure 2, Figure 1. a) Base-pairing scheme of spin labels Ç and with G. dR is 2’-deoxyribose. b) Structure of an abasic site in DNA.

Site-Directed Nitroxide Spin Labeling of Biopolymers

The application of electron paramagnetic resonance (EPR) spectroscopy to the study of biopolymer structure and dynamics has seen rapid growth in the last decade. In addition to advances in instrumentation – in particular, the development of high-field spectrometers and pulsed-EPR methods – spin-labeling techniques have evolved. Nitroxide spin labels can now routinely be incorporated at selected sites to interrogate how structure and dynamics at specific locations relate to biopolymer function. Furthermore, spin labels with improved properties have emerged, in particular, rigid labels that yield more accurate distance measurements, give information about orientation, and faithfully report site-specific dynamics. This review recounts how the three main approaches for site-directed spin labeling of biopolymers, namely, postsynthetic labeling, labeling during biopolymer synthesis, and noncovalent labeling, have been used to label proteins as well as nucleic acids.

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.

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 C3-spacer (C3) 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.

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.

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 for EPR Studies of Nucleic Acids

Electron paramagnetic resonance (EPR) spectroscopy has emerged as a valuable technique to study the structure and dynamics of nucleic acids and their complexes with other biomolecules. EPR studies require incorporation of stable free radicals (spin labels), usually aminoxyl radicals (nitroxides), at specific sites in the nucleic acids using site-directed spin labeling (SDSL). In addition to the advancement of EPR instrumentation and pulsed EPR techniques, new strategies for SDSL have emerged, in particular, use of click chemistry, biopolymer catalysis, and noncovalent labeling. Furthermore, tailor-made spin labels with improved stability and spectroscopic properties have evolved, such as rigid spin labels that allow determination of accurate distances in addition to orientations between two spin labels. This chapter gives an overview of nucleic acids spin labeling using the three main strategies of SDSL, namely spin labeling during oligonucleotide synthesis, post-synthetic-, and noncovalent labeling. The spin-labeling methods have been categorized according to the labeling site.

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

Abstract 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.

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.The DIR2s RNA aptamer activates the fluorescence of cyanine dyes allowing detection of two well-resolved emission colors. Here authors solve the crystal structures of the apo and OTB-SO3 fluorophore-bound DIR2s and show how the fluorophore ligand is bound.