In vitro selection of phosphorothiolated aptamers

A pool of RNA molecules that contained exclusively phosphorothioate internucleoside linkages was used as a starting point for the selection of aptamers that bind to basic fibroblast growth factor (bFGF), and appear to act as heparin mimics.     

In vitro selection of bacteriophage phi29 prohead RNA aptamers for prohead binding

Prohead RNA (pRNA) of the Bacillus subtilis bacteriophage phi29 is needed for in vitro packaging of DNA-gene product 3 (DNA-gp3). Residues 22-84 of the 174-base pRNA bind the portal vertex of the prohead, the site of DNA packaging. To define the nucleotides of pRNA needed for prohead binding and DNA-gp3 packaging and to seek biologically active variants of pRNA, segments of pRNA were randomized to obtain vast repertoires of RNA molecules. RNA aptamers, ligands best suited for prohead binding, were obtained by multiple rounds of in vitro selection. Evolution of pRNA aptamers was followed by a competition binding assay and nucleotide sequencing, and mutants were tested for DNA-gp3 packaging. Aptamers selected following randomization of the E stem and loop and a part of the C-E loop that were active in DNA-gp3 packaging were invariably wild-type. DNA-gp3 packaging activity also required nucleotides G82 and G83 that form base pairs intermolecularly with C47 and C48 to produce a novel hexameric oligomer of pRNA. The only mutant aptamers that retained full DNA-gp3 packaging activity showed changes of the U residues at positions 81, 84, and 85 of the D loop. Thus, the in vitro selections essentially recapitulated the natural evolution of pRNA.     

Pregnanes that bind to the digitalis receptor

Steroid binding domains of Na,K-ATPase and the nuclear hormone receptors share amino acid sequence homologies. In a ouabain radioligand assay, the potencies of series of planar or bent steroid moieties suggest that the domain in Na,K-ATPase can accommodate compounds with a planar configuration. The A/B -cis, C/D-cis steroid configuration in the cardenolides, in conjunction with appropriate substituents at the 3 and 17 positions, may represent a fortuitous "fit" for the exogenous compounds. It remains to be determined if the putative physiological digitalis-like substance is a member of a hormonal steroid family, an endogenous ouabain-like compound or both.     

In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443-473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.     

In vitro selection of dopamine RNA ligands

RNA aptamers that specifically bind dopamine have been isolated by in vitro selection from a pool of 3.4 x 10(14) different RNA molecules. One aptamer (dopa2), which dominated the selected pool, has been characterized and binds to the dopamine affinity column with a dissociation constant of 2.8 microM. The specificity of binding has been determined by studying binding properties of a number of dopamine-related molecules, showing that the interaction with the RNA might be mediated by the hydroxyl group at position 3 and the proximal aliphatic chain in the dopamine molecule. The binding domain was initially localized by boundary experiments. Further definition of the dopamine binding site was obtained by secondary selection on a pool of sequences derived from a partial randomization of the dopa2 molecule. Sequence comparison of a large panel of selected variants revealed a structural consensus motif among the active aptamers. The dopamine binding pocket is built up by a tertiary interaction between two stem and loop motifs, creating a stable framework in which five invariant nucleotides are precisely arrayed. Minimal active sequence and key nucleotides have been confirmed by the design of small functional aptamers and mutational analysis. Enzymatic probing suggests that the RNA might undergo a conformational change upon ligand binding that stabilizes the proposed tertiary structure.     

In vitro selection of a 7-methyl-guanosine binding RNA that inhibits translation of capped mRNA molecules

Using systematic evolution of ligands by exponential enrichment (SELEX), an RNA molecule was isolated that displays a 1,000-fold higher affinity for guanosine residues that carry an N-7 methyl group than for nonmethylated guanosine residues. The methylated guanosine residue closely resembles the 5' terminal cap structure present on all eukaryotic mRNA molecules. The cap-binding RNA specifically inhibited the translation of capped but not uncapped mRNA molecules in cell-free lysates prepared from either human HeLa cells or from Saccharomyces cerevisiae. These findings indicate that the cap-binding RNA will also be useful in studies of other cap-dependent processes such as pre-mRNA splicing and nucleocytoplasmic mRNA transport.     

In vitro selection analysis of trans-acting HDV ribozyme

In order to identify the functional structure as well as new active variants of the trans-acting genomic ribozyme of human hepatitis delta virus (HDV), we applied an in vitro selection procedure. After 10 generations, a randomized pool of trans-acting ribozymes accumulated in which the secondary structure of each ribozyme confirmed to the pseudoknot model and important bases in single-stranded regions were all conserved. We were surprised that mutated ribozymes derived from genomic sequence were changed to anti-genomic-like sequences. Further investigations of the most active variant confirmed that each mutated base was the most appropriate nucleotide at every position of HDV ribozyme.     

In vitro selection of the Naegleria GIR1 ribozyme identifies three base changes that dramatically improve activity

NanGIR1 is a member of a new class of group I ribozymes whose putative biological function is site-specific hydrolysis at an internal processing site (IPS). We have previously shown that NanGIR1 requires 1 M KCl for maximal activity, which is nevertheless slow (0.03 min(-1)). We used in vitro selection and an RNA pool with approximately nine mutations per molecule to select for faster hydrolysis at the IPS in 100 mM KCl. After eight rounds of selection, GIR1 variants were isolated that catalyzed hydrolysis at 300-fold greater rates than NanGIR1 RNA. Although not required by the selection, many of the resultant RNAs had increased thermal stability relative to the parent RNA, and had a more compact structure as evidenced by their faster migration in native gels. Although a wide spectrum of mutations was found in generation 8 clones, only two mutations, U149C and U153C, were common to greater than 95% of the molecules. These and one other mutation, G32A, are sufficient to increase activity 50-fold. All three mutations lie within or proximal to the P15 pseudoknot, a structural signature of GIR1 RNAs that was previously shown to be important for catalytic activity. Overall, our findings show that variants of the Naegleria GIR1 ribozyme with dramatically improved activity lie very close to the natural GIR1 in sequence space. Furthermore, the selection for higher activity appeared to select for increased structural stability.     

DNA ligands that bind tightly and selectively to cellobiose

Cell surface oligosaccharides have been shown to play essential biological roles in such diverse biological phenomena as cellular adhesion, molecular recognition, and inflammatory response. The development of high-affinity ligands capable of selectively recognizing a variety of small motifs in different oligosaccharides would be of significant interest as experimental and diagnostic tools. As a step toward this goal we have developed DNA ligands that recognize the disaccharide cellobiose, whether in soluble form or as the repeating unit of the polymer, cellulose. These DNA "aptamers" bind with high selectivity to cellobiose with little or no affinity for the related disaccharides lactose, maltose, and gentiobiose. Thus, the DNA ligands can discriminate sugar epimers, anomers, and disaccharide linkages.     

In vitro synthesis of the unit that links teichoic acid to peptidoglycan

The role of cytidine diphosphate (CDP)-glycerol in gram-positive bacteria whose walls lack poly(glycerol phosphate) was investigated. Membrane preparations from Staphylococcus aureus H, Bacillus subtilis W23, and Micrococcus sp. 2102 catalyzed the incorporation of glycerol phosphate residues from radioactive CDP-glycerol into a water-soluble polymer. In toluenized cells of Micrococcus sp. 2102, some of this product became linked to the wall. In each case, maximum incorporation of glycerol phosphate residues required the presence of the nucleotide precursors of wall teichoic acid and of uridine diphosphate-N-acetylglucosamine. In membrane preparations capable of synthesizing peptidoglycan, vancomycin caused a decrease in the incorporation of isotope from CDP-glycerol into polymer. Synthesis of the poly (glycerol phosphate) unit thus depended at an early stage on the concomitant synthesis of wall teichoic acid and later on the synthesis of peptidoglycan. It is concluded that CDP-glycerol is the biosynthetic precursor of the tri(glycerol phosphate) linkage unit between teichoic acid and peptidoglycan that has recently been characterized in S. aureus H.     

In vitro selection of resistance to four beta-lactams and azithromycin in Streptococcus pneumoniae

Selection of resistance to amoxicillin (with or without clavulanate), cefaclor, cefuroxime, and azithromycin among six penicillin G- and azithromycin-susceptible pneumococcal strains and among four strains with intermediate penicillin sensitivities (azithromycin MICs, 0.125 to 4 microg/ml) was studied by performing 50 sequential subcultures in medium with sub-MICs of these antimicrobial agents. For only one of the six penicillin-susceptible strains did subculturing in medium with amoxicillin (with or without clavulanate) lead to an increased MIC, with the MIC rising from 0.008 to 0.125 microg/ml. Five of the six penicillin-susceptible strains showed increased azithromycin MICs (0.5 to >256.0 microg/ml) after 17 to 45 subcultures. Subculturing in medium with cefaclor did not affect the cefaclor MICs of three strains but and led to increased cefaclor MICs (from 0.5 to 2.0 to 4.0 microg/ml) for three of the six strains, with MICs of other beta-lactams rising 1 to 3 twofold dilutions. Subculturing in cefuroxime led to increased cefuroxime MICs (from 0.03 to 0.06 microg/ml to 0.125 to 0.5 microg/ml) for all six strains without significantly altering the MICs of other beta-lactams, except for one strain, which developed an increased cefaclor MIC. Subculturing in azithromycin did not affect beta-lactam MICs. Subculturing of the four strains with decreased penicillin susceptibility in amoxicillin (with or without clavulanate) or cefuroxime did not select for beta-lactam resistance. Subculturing of one strain in cefaclor led to an increase in MIC from 0.5 to 2.0 microg/ml after 19 passages. In contrast to strains that were initially azithromycin susceptible, which required >10 subcultures for resistance selection, three of four strains with azithromycin MICs of 0.125 to 4.0 microg/ml showed increased MICs after 7 to 13 passages, with the MICs increasing to 16 to 32 microg/ml. All azithromycin-resistant strains were clarithromycin resistant. With the exception of strains that contained mefE at the onset, no strains that developed resistance to azithromycin contained ermB or mefE, genes that have been found in macrolide-resistant pneumococci obtained from clinic patients.     

In vitro selection of RNA lectins: using combinatorial chemistry to interpret ribozyme evolution

BACKGROUND: It has been hypothesized that the fact that both ribosomal RNA and the group I intron can bind to aminoglycoside antibiotics implies that these RNAs are evolutionarily related. This hypothesis requires the assumption that there are relatively few ways for RNA molecules to form aminoglycoside-binding sites. RESULTS: We have used in vitro selection to determine the diversity of aminoglycoside-binding sites that can be formed by RNA molecules. We have generated RNA 'lectins' that can bind aminoglycosides tightly and specifically. Sequence analysis indicates that there are many different ways to form tight and specific aminoglycoside binding sites. These artificially selected binding sites are functionally similar to those that have arisen from natural selection. CONCLUSIONS: Our results suggest that the presence of aminoglycoside-binding sites on RNA molecules may not be a useful trait for determining evolutionary relatedness. Instead, the fact that RNA molecules can bind these 'low molecular-weight effectors' may indicate that natural products such as aminoglycosides have evolved to exploit sequence- and structure-specific recognition of nucleic acids, in much the same way that lexitropsins have been designed by chemists to recognise specific nucleic acid sequences.     

In vitro selection of self-cleaving RNAs with a low pH optimum

RNAs that undergo a rapid site-specific cleavage at low pH have been selected by in vitro selection (the SELEX process). The cleavage does not require the addition of any divalent metal ions, and is in fact inhibited by divalent metal ions, spermine, or high concentrations of monovalent metal ions. This low pH catalyzed cleavage results in a 2',3'-cyclic phosphate at the 3' end and a free hydroxyl at the 5' end. The reaction proceeds with a calculated rate of 1.1 min-1 at room temperature in cacodylate buffer at pH 5.0. The rate of cleavage is dependent on the pH and shows an optimum around pH 4.0. The rate constant is independent of RNA concentration, indicating to an intramolecular reaction. Autocatalytic cleavage at low pH, in the absence of a metal ion requirement, adds to the reaction possibilities that may have existed on the prebiotic earth.     

Differential response of human melanoma and Ehrlich ascites cells in vitro to the ribosome-inactivating protein luffin

OBJECTIVE: To analyze the short- and medium-term results of the Burch-like urethropexy with bone anchors in the treatment of genuine stress urinary incontinence. METHODS: We performed the conventional Burch technique which was modified with the use of 4 bone anchors for bony fixation. Forty-four female patients with genuine stress urinary incontinence were operated on from November, 1995 to November, 1997. RESULTS: The patients had a bladder catheter indwelling for 4 to 9 days and only 3 of them required intermittent catheterization during two months. All patients recovered spontaneous micturition. The postoperative urinary continence was 93% at a mean follow-up of 11 months. CONCLUSIONS: Although our initial results seem encouraging, a continuous and objective follow-up is warranted to assess the long-term efficacy of this technique.     

Two proteins of the Dictyostelium spore coat bind to cellulose in vitro

The spore coat of Dictyostelium contains nine different proteins and cellulose. Interactions between protein and cellulose were investigated using an in vitro binding assay. Proteins extracted from coats with urea and 2-mercaptoethanol could, after removal of urea by gel filtration, efficiently bind to particles of cellulose (Avicel), but not Sephadex or Sepharose. Two proteins, SP85 and SP35, were enriched in the reconstitution, and they retained their cellulose binding activities after purification by ion exchange chromatography under denaturing conditions to suppress protein--protein interactions. Neither protein exhibited cellulase activity, though under certain conditions SP85 copurified with a cellulase activity which appeared after germination. Amino acid sequencing indicated that SP85 and SP35 are encoded by the previously described pspB and psvA genes. This was confirmed for SP85 by showing that natural M(r) polymorphisms correlated with changes in the number of tetrapeptide-encoding sequence repeats in pspB. Using PCR to reconstruct missing elements from the recombinogenic middle region of pspB, SP85 was shown to consist of three sequence domains separated by two groups of the tetrapeptide repeats. Expression of partial pspB cDNAs in Escherichia coli showed that cellulose-binding activity resided in the Cys-rich COOH-terminal domain of SP85. This cellulose-binding activity can explain SP85's ultrastructural colocalization with cellulose in vivo. Amino acid composition and antibody binding data showed that SP35 is derived from the Cys-rich N-terminal region of the previously described psvA protein. SP85 and SP35 may link other proteins to cellulose during coat assembly and germination.     

Oligonucleotide inhibitors of human thrombin that bind distinct epitopes

Thrombin, a multifunctional serine protease, recognizes multiple macromolecular substrates and plays a key role in both procoagulant and anticoagulant functions. The substrate specificity of thrombin involves two electropositive surfaces, the fibrinogen-recognition and heparin-binding exosites. The SELEX process is a powerful combinatorial methodology for identifying high-affinity oligonucleotide ligands to any desired target. The SELEX process has been used to isolate single-stranded DNA ligands to human thrombin. Here, a 29-nucleotide single-stranded DNA ligand to human thrombin, designated 60-18[29], with a Kd of approximately 0.5 nM is described. DNA 60-18[29] inhibits thrombin-catalyzed fibrin clot formation in vitro. Previously described DNA ligands bind the fibrinogen-recognition exosite, while competition and photocrosslinking experiments indicate that the DNA ligand 60-18[29] binds the heparin-binding exosite. DNA 60-18[29] is a quadruplex/duplex with a 15-nucleotide "core" sequence that has striking similarity to previously described DNA ligands to thrombin, but binds with 20 to 50-fold higher affinity. The 15-nucleotide core sequence has eight highly conserved guanine residues and forms a G-quadruplex structure. A single nucleotide within the G-quadruplex structure can direct the DNA to a distinct epitope. Additional sequence information in the duplex regions of ligand 60-18[29] contribute to greater stability and affinity of binding to thrombin. A low-resolution model for the interaction of DNA 60-18[29] to human thrombin has been proposed.     

Helicobacter mustelae and Helicobacter pylori bind to common lipid receptors in vitro

Helicobacter pylori is a recently recognized human pathogen causing chronic-active gastritis in association with duodenal ulcers and gastric cancer. Helicobacter mustelae is a closely related bacterium with similar biochemical and morphologic characteristics. H. mustelae infection of antral and fundic mucosa in adult ferrets causes chronic gastritis. An essential virulence property of both Helicobacter species is bacterial adhesion to mucosal surfaces. The aim of this study was to determine whether H. mustelae binds to the same lipids shown previously to be receptors for H. pylori adhesion in vitro. By using thin-layer chromatography overlay and a receptor-based enzyme-linked immunosorbent assay, H. mustelae was found to bind the same receptor lipids as H. pylori, namely, phosphatidylethanolamine and gangliotetraosylceramide. In addition, both H. pylori and H. mustelae bound to a deacylplasmalogen phosphatidylethanolamine. In contrast to H. pylori, H. mustelae binding to receptors was unaffected by motility or viability. Murine monoclonal and bovine polyclonal antibodies against exoenzyme S, and exoenzyme S itself (from Pseudomonas aeruginosa), inhibited binding of H. mustelae to phosphatidylethanolamine and gangliotetraosylceramide. These findings show that H. mustelae binds in vitro to the same lipid receptors as H. pylori and suggest that the adhesion of H. mustelae to such species is mediated by preformed, surface-exposed adhesins which include an exoenzyme S-like protein.     

Predicting peptides that bind to MHC molecules using supervised learning of hidden Markov models

Cells in the lateral hypothalamus and in the arcuate nucleus play prominent roles in the central control of food intake; however, a neurochemical link connecting these potential components of a hypothalamic circuitry regulating energy metabolism remains to be established. In the present study, the topographical relationship between cells expressing mRNAs encoding melanin-concentrating hormone and the newly discovered neuropeptide family hypocretins/orexins was studied in the rat and mouse lateral hypothalamus by using double-labeling in situ hybridization. Cells expressing the two mRNAs formed completely distinct populations, with hypocretin/orexin cells located primarily perifornically and in the magnocellular lateral hypothalamic nucleus; melanin-concentrating hormone cells extended in a wider area both laterally and periventricularly and appeared to partly surround the hypocretin/orexin population. In the arcuate nucleus, cells expressing neuropeptide Y and agouti gene-related protein were studied by routine fluorescence and/or confocal microscopy immunohistochemistry. Double staining demonstrated that a large proportion of the neuropeptide Y-positive cell bodies in this nucleus also contained agouti gene-related protein-like immunoreactivity. Moreover, these two peptides also coexisted in nerve terminals surrounding and in close relationship to perikarya and processes of both hypocretin/orexin- and melanin-concentrating hormone-immunoreactive cells in the lateral hypothalamus, whereby the former appeared to receive a more dense innervation. These results thus provide evidence for an arcuate-lateral hypothalamic neuropeptide Y/agouti gene-related protein pathway. Furthermore, the results implicate hypocretin/orexin and melanin-concentrating hormone-expressing cells as downstream targets in neuropeptide Y-induced feeding.