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Boron neutron capture therapy (BNCT), an experimental treatment for certain cancers, destroys only cells near the boron; however, there is a need to develop highly specific delivery agents. As nucleic acid aptamers recognize specific molecular targets, we investigated the influence of boronated nucleotide analogs on RNA function and on the systematic evolution of ligands by exponential enrichment (SELEX) process. Substitution of guanosine 5'-(alpha-P-borano) triphosphate (bG) for GTP or uridine 5'-(alpha-P-borano) triphosphate (bU) for UTP in several known aptamers diminished or eliminated target recognition by those RNAs. Specifically, ATP-binding aptamers containing the zeta-fold, which appears in several selections for adenosine aptamers, became inactive upon bG substitution but were only moderately affected by bU substitution. Selections were carried out using the bG or bU analogs with C8-linked ATP agarose as the binding target. The selections with bU and normal NTP yielded some zeta-fold aptamers, while the bG selection yielded none of this type. Non-zeta aptamers from bU and bG populations tolerated the borano substitution and many required it. The borano nucleotide requirement is specific; bU could not be used in bG-dependent aptamers nor vice versa. The borano group plays an essential role, as yet undefined, in target recognition or RNA structure. We conclude that the bG and bU nucleotides are fully compatible with SELEX, and that these analogs could be used to make boronated aptamers as therapeutics for BNCT.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is employed to detect the presence of the bacteriological sex factor, a trait that allows some bacteria to transfer genetic information to others by means of conjugation. The principal advantage of the method is its speed. Twenty different strains of E. coli were analyzed and the results were consistent with previously known genetic information. The sex factor could be transferred from one strain to another and the outcome verified by mass spectrometry. © 1998 John Wiley & Sons, Ltd.

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.

Flavin adenine dinucleotide (FAD) is one of the primary cofactors in biological redox reactions. Designing cofactor-dependent redox ribozymes could benefit from studies of new RNA-cofactor complexes, as would our understanding of ribozyme evolution during an RNA World. We have therefore used the SELEX method to identify RNA aptamers that recognize FAD. Functional analysis of mutant aptamers, S1 nuclease probing, and comparative sequence analysis identified a simple, 45 nt helical structure with several internal bulges as the core-binding element. These aptamers recognize with high specificity the isoalloxazine nucleus of FAD but do not distinguish FAD from FADH(2), nor are they removed from an FAD resin with UMP (which shares a pattern of hydrogen bond donors and acceptors along one face). Thus, these aptamers are structurally and functionally distinct from previously identified FMN and riboflavin aptamers. Circular dichroism data suggest a conformational change in the RNA upon FAD binding. These aptamers require magnesium and are active across a wide pH range (4.5-8.9). Since general acid-base catalysis plays a role in some flavin-dependent redox reaction mechanisms, these aptamers may be particularly well-suited to the design of new redox ribozymes.

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.

Screening new drugs is a costly and time-consuming process. Identifying new targets for existing therapeutics is often a particularly effective avenue for drug development. We have investigated whether in vitro selection can be used for target acquisition. Aminoglycoside antibiotics are known to bind to and inactivate functional natural nucleic acids, such as ribosomal RNA. As an example of how new targets for aminoglycosides could be identified, a lividomycin aptamer was iteratively isolated from a random sequence pool. The consensus sequence of this and other anti-aminoglycoside aptamers was used as the basis for a comprehensive search of natural sequence databases. Surprisingly, a high degree of similarity was found between aptamers and genomic sequences from a variety of organisms. While many of the similarities found are in regions of unknown or nonessential function, some of the sequences are found in critical genes in pathogenic organisms.

It is generally believed that the earliest progenitor of modern life was a set of chemical shapes or reactions that could reproduce itself. Although it is convenient to hypothesize that life began with a nucleic acid such as RNA, there is no a priori reason to believe that the earliest replicators were composed of compounds identical to those found in contemporary organisms. The difficulties inherent in forming nucleotide and oligonucleotide precursors under prebiotic conditions (Pace, 1991) have led many authors to propose that the earliest replicators may have been ‘unnatural’ nucleic acids that contained modified backbones or internucleotide linkages and replicated via complementary chemical shapes such as Watson-Crick base pairings (reviewed in Joyce, 1989; Ellington, 1993). However, while Watson-Crick style complementarity is clearly well-suited to copying chemical information, it is not necessary to envisage life. Wachtershauser (1988) has proposed a scenario in which the earliest replicators were chemical cycles, and Cairns-Smith (1982) has suggested that the first living systems were crystalline matrices, such as clays, that could grow and diversify. These non-Watson-Crick replicators were gradually replaced by nucleic acids in a ‘genetic takeover’ that led to modern life.

Visible wavelength ligand-to-metal (LMCT) activated N2 release from tris(3-hydroxy-1,2,3-benzotriazine-4(3H)-one]iron(III) produces localized ligand radical intermediates capable of cleaving DNA and represents a new chemical approach to photonuclease design for biological applications.

Bis(9-diazo-4,5-diazafluorene)copper(II) nitrate is an effective DNA photocleaving agent under anaerobic conditions using visible light and represents a potential model for the action of Kinamycin antitumor antibiotics.