Charles Wilson

Isolation and characterization of fluorophore-binding RNA aptamers

BACKGROUNDnIn vitro selection has been shown previously to be a powerful method for isolating nucleic acids with specific ligand-binding functions (aptamers). Given this capacity, we have sought to isolate RNA motifs that can confer fluorescent labeling to tagged RNA transcripts, potentially allowing in vivo detection and in vitro spectroscopic analysis of RNAs.nnnRESULTSnTwo aptamers that recognize the fluorophore sulforhodamine B were isolated by the in vitro selection process. An unusually large motif of approximately 60 nucleotides is responsible for binding in one RNA (SRB-2). This motif consists of a three-way helical junction with two large, highly conserved unpaired regions. Phosphorothioate mapping with an iodoacetamide-tagged form of the ligand shows that these two regions make close contacts with the fluorophore, suggesting that the two loops combine to form separate halves of a binding pocket. The aptamer binds the fluorophore with high affinity, recognizing both the planar aromatic ring system and a negatively charged sulfonate, a rare example of anion recognition by RNA. An aptamer (FB-1) that specifically binds fluorescein has also been isolated by mutagenesis of a sulforhodamine aptamer followed by re-selection. In a simple in vitro test, SRB-2 and FB-1 have been shown to discriminate between sulforhodamine and fluorescein, specifically localizing each fluorophore to beads tagged with the corresponding aptamer.nnnCONCLUSIONSnIn addition to serving as a model system for understanding the basis of RNA folding and function, these experiments demonstrate potential applications for the aptamers in transcript double labeling or fluorescence resonance energy transfer studies.

Inducible regulation of the S. cerevisiae cell cycle mediated by an RNA aptamer-ligand complex.

Previous studies have shown that the introduction of a ligand-binding RNA (aptamer) into the 5-UTR of an mRNA can confer regulated expression of both prokaryotic and eukaryotic reporter genes. The current report shows that aptamer insertion into the 5-UTR of a cyclin transcript in S. cerevisiae renders cell-cycle control dependent upon the presence or absence of the target ligand. A malachite green binding motif, defined by an asymmetric internal loop flanked by short RNA helices, was inserted immediately upstream of the CLB2 start codon. Progression through the cell cycle is dramatically slowed and elongated bud morphology develops when tetramethylrosamine (a fluorescent malachite green analogue) is added to the aptamer-containing strain. Quantification of CLB2 expression at the RNA and protein levels by RT-PCR and Western blot analysis, respectively, demonstrates that the aptamer ligand regulates transcript translatability rather than stability. One-dimensional NMR spectroscopy shows that the malachite green binding aptamer undergoes a dramatic ligand-dependent change in structure with many nucleotides folding to adopt a well-defined conformation. These results are consistent with a model in which translational initiation is blocked by ligand-induced conformational changes in the 5-UTR.

Isolation of a fluorophore-specific DNA aptamer with weak redox activity

BACKGROUNDnIn vitro selection experiments with pools of random-sequence nucleic acids have been used extensively to isolate molecules capable of binding specific ligands and catalyzing self-modification reactions.nnnRESULTSnIn vitro selection from a random pool of single-stranded DNAs has been used to isolate molecules capable of recognizing the fluorophore sulforhodamine B with high affinity. When assayed for the ability to promote an oxidation reaction using the reduced form of a related fluorophore, dihydrotetramethylrosamine, a number of selected clones show low levels of catalytic activity. Chemical modification and site-directed mutagenesis experiments have been used to probe the structural requirements for fluorophore binding. The aptamer recognizes its ligand with relatively high affinity and is also capable of binding related molecules that share extended aromatic rings and negatively charged functional groups.nnnCONCLUSIONSnA guanosine-rich single-stranded DNA is capable of binding fluorophores with relatively high affinity and of weakly promoting a multiple-turnover reaction. A simple motif consisting of a three-tiered G-quartet stacked upon a standard Watson-Crick duplex appears to be responsible for this activity. The corresponding sequence might provide a useful starting point for the evolution of novel, improved deoxyribozymes that generate fluorescent signals by promoting multiple-turnover reactions.

Recognition of Planar and Nonplanar Ligands in the Malachite Green–RNA Aptamer Complex

Ribonucleic acids are an attractive drug target owing to their central role in many pathological processes. Notwithstanding this potential, RNA has only rarely been successfully targeted with novel drugs. The difficulty of targeting RNA is at least in part due to the unusual mode of binding found in most small‐molecule–RNA complexes: the ligand binding pocket of the RNA is largely unstructured in the absence of ligand and forms a defined structure only with the ligand acting as scaffold for folding. Moreover, electrostatic interactions between RNA and ligand can also induce significant changes in the ligand structure due to the polyanionic nature of the RNA. Aptamers are ideal model systems to study these kinds of interactions owing to their small size and the ease with which they can be evolved to recognize a large variety of different ligands. Here we present the solution structure of an RNA aptamer that binds triphenyl dyes in complex with malachite green and compare it with a previously determined crystal structure of a complex formed with tetramethylrosamine. The structures illustrate how the same RNA binding pocket can adapt to accommodate both planar and nonplanar ligands. Binding studies with single‐ and double‐substitution mutant aptamers are used to correlate three‐dimensional structure with complex stability. The two RNA–ligand complex structures allow a discussion of structural changes that have been observed in the ligand in the context of the overall complex structure. Base pairing and stacking interactions within the RNA fold the phosphate backbone into a structure that results in an asymmetric charge distribution within the binding pocket that forces the ligand to adapt through a redistribution of the positive partial charge.

A water channel in the core of the vitamin B12 RNA aptamer

BACKGROUNDnThe 3.0 A crystal structure of the vitamin B(12) RNA aptamer revealed an unusual tertiary structure that is rich in novel RNA structural motifs. Important details of the interactions that stabilize noncanonical base pairing and the role of solvent in the structure were not apparent owing to the limited resolution.nnnRESULTSnThe structure of the vitamin B(12) RNA aptamer in complex with its ligand has been determined at 2.3 A resolution by X-ray crystallography. The crystallographic asymmetric unit contains five independent copies of the aptamer-vitamin B(12) complex, making it possible to accurately define well-conserved features. The core of the aptamer contains an unusual water-filled channel that is buried between the three strands of an RNA triplex. Well-ordered water molecules positioned within this channel form bridging hydrogen bonds and stabilize planar base triples that otherwise lack significant direct base-base contacts. The water channel terminates at the interface between the RNA and the bound ligand, leaving a pair of water molecules appropriately positioned to hydrogen bond with the highly polarized cyanide nitrogen of vitamin B(12). Analysis of the general solvation patterns for each nucleotide suggests that water molecules are not precisely positioned, as observed in previous RNA duplex structures, but instead might adjust in response to the varying local environment. Unusual intermolecular base pairing contributes to the formation of three different dimerization contacts that drive formation of the crystal lattice.nnnCONCLUSIONSnThe structure demonstrates the important role of water molecules and noncanonical base pairing in driving the formation of RNA tertiary structure and facilitating specific interactions of RNAs with other molecules.

Preliminary characterization of crystals of an in vitro evolved cyanocobalamin (vitamin B12) binding RNA.

A 35-nucleotide pseudoknot that binds vitamin B12 been isolated using systematic evolution of ligands by exponential enrichment (SELEX). Affinity chromatography was used to purify functional, properly folded molecules and the hanging-drop vapor-diffusion method was used to crystallize this aptamer. Two crystal forms have been obtained and the preliminary crystallographic characterization is reported here. Both crystal forms (space groups I222 or I212121 and C2221) diffract to 2.9 A and should prove sufficient for structure determination.

Archemix: drug discovery innovation based on evolutionary nucleic acid technology platforms

Abstract Archemix, develops aptamers and riboreporters tm (allosteric ribozymes that couple recognition to detection) for therapeutics and drug discovery. Aptamers and riboreporters tm recognize drugs, metabolites and proteins, and function in multiple formats, including chip, solution and cellular settings. Using novel nucleic acid platforms, Archemix builds mechanism-based drug discovery programs around virtually any target.

Preliminary crystallographic characterization of an in vitro evolved biotin-binding RNA pseudoknot

A biotin-binding RNA pseudoknot developed through in vitro selection has been crystallized using the hanging-drop vapor-phase diffusion method. The X-ray diffraction data indicate that the crystals belong to the space group P4222 with unit-cell parameters a = b = 55.2, c = 62.7 A and alpha = beta = gamma = 90 degrees. The crystals are 120 x 80 x 40 microm and VM = 2.17 A3 Da-1. The crystals diffract to 2.8 A.