RNase J1 endonuclease activity as a probe of RNA secondary structure

Reliable determination of RNA secondary structure depends on both computer algorithms and experimental probing of nucleotides in single- or double-stranded conformation. Here we describe the exploitation of the endonucleolytic activity of the Bacillus subtilis enzyme RNase J1 as a probe of RNA structure. RNase J1 cleaves in single-stranded regions and, in vitro at least, the enzyme has relatively relaxed nucleotide specificity. We confirmed the feasibility of the approach on an RNA of known structure, B. subtilis tRNA^Thr. We then used RNase J1 to solve the secondary structure of the 5′ end of the hbs mRNA. Finally, we showed that RNase J1 can also be used in footprinting experiments by probing the interaction between the 30S ribosomal subunit and the Shine–Dalgarno element of the hbs mRNA.     

Measuring the thermodynamics of RNA secondary structure formation

The thermodynamics of RNA secondary structure formation in small model systems provides a database for predicting RNA structure from sequence. Methods for making these measurements are reviewed with emphasis on optical methods and treatment of experimental errors. Analysis of experimental results in terms of simple nearest-neighbor models is presented. Some measured sequence dependences of non-Watson-Crick motifs are discussed. © 1998 John Wiley & Sons, Inc. Biopoly 44: 309–319, 1997     

Terbium as a solid-state probe for RNA

This paper continues previous work on the analysis of nucleic acid-terbium complexes in the solid state. The fluorescence excitation and emission spectra of the RNA-terbium(III) complex is reported. The fluorescence excitation and emission spectra of both the RNA-terbium(III) and DNA-terbium(III) complexes as trapped on millipore filters is reported. One hundred percent of the DNA combined with terbium was trapped on millipore filters. Deoxyribonucleic acid was recovered from DNA-terbium(III) complexes trapped on millipore filters using SDS-extraction. Energy transfer was shown to occur from the bases in nucleic acids to the terbium ion, whereas the actual binding of terbium to nucleic acids was due to phosphate groups. The relative fluorescence of homopolyribonucleotide-terbium complexes showed that the guanine moiety was responsible for most of the observed fluorescence. Binding studies showed an equal affinity of radioactive terbium for all the homopolyribonucleotides. The fluorescence of solid-state DNA and RNA terbium complexes was used to measure picomole quantities of DNA or RNA.     

Tb(III) as a fluorescent probe for the structure of bovine serum albumin

Tb(III) was used as a fluorescent probe in the study of the calcium-binding sites on Bovine Serum Albumin (BSA). The fluorescence of Tb(III) is enhanced markedly when bound to BSA and nonradiative energy transfer between two fluorescent tryptophan(Trp) residues and Tb(III) bound to calcium-binding sites on BSA occurred. Experimental results show that the major groups in BSA bound to metal ion are the carboxyl side groups of glutamic acid (Glu) and aspartic acid (Asp). The average distance between the bound Tb(III) and the two tryptophan residues in BSA calculated by a F?ster dipole-dipole nonradiative energy transfer mechanism is 1.48 nm.     

Quinolizinium as a new fluorescent lysosomotropic probe

We have synthesized a collection of quinolizinium fluorescent dyes for the purpose of cell imaging. Preliminary biological studies in human U2OS osteosarcoma cancer cells have shown that different functional groups appended to the cationic quinolizinium scaffold efficiently modulate photophysical properties but also cellular distribution. While quinolizinium probes are known nuclear staining reagents, we have identified a particular quinolizinium derivative salt that targets the lysosomal compartment. This finding raises the question of predictability of specific organelle targeting from structural features of small molecules.     

Study of the secondary and tertiary structure of phage MS2 RNA using nucleases and fluorescent dyes specific for secondary structure

The binding of ethidium bromide (EtBr) and acridine orange (AO) to RNA in native state or after hydrolysis by S1 and SV nucleases that specifically split single-stranded and double-stranded segments was studied. Nuclease S1 hydrolysis of RNA does not increase the number of EtBr strong binding sites, Tm and hyperchromic effect being also unchanged. Hydrolysis by double-stranded segments accessible to EtBr is followed by the diminishing of Tm and hyperchromism. A supposition is put forward that the main role in stabilization of the RNA tertiary structure is played by double-stranded segments arranged so that some of them are hidden and do not interact with dyes. One of the possible models may be parallel oriented intramolecular "hair-pins" forming compact "rod-like" structures.     

Terbium as a fluorescent probe for DNA and chromatin.

Terbium reacted with DNA and chromatin to form a complex in which terbium acted as a sensitive fluorescent probe. By measuring the narrow-line emission of Tb-3+ when DNA is selectively excited, the relative amount of Tb-3+ bound to the DNA can be calculated. Terbium was bound to DNA until one Tb-3+ was present for each phosphate group. After this point no more terbium was bound. TbCl3 was bound to chromatin in a linear manner until approximately 0.48 TbCl3 was added for each phosphate group in the chromatin-DNA solution. From these data it appears that 52% of the phosphate groups in chromatin were unavailable for binding. The binding of Tb-3+ to DNA can be reversed by prolonged dialysis against 0.5 M NaCl and chelating agents. The terbium ion is ideal in that it binds DNA tight enough so that completion of the reaction can be assumed but loose enough so that it can be removed by gentle means. Low concentrations of salt (up to 2 mM NaCl) enhance the quantum efficiency. Below pH 3 and above pH 7 the DNA-terbium complex will not form. Between pH 3 and pH 7 the quantum efficiency of the DNA terbium complex increases from either pH to a maximum at pH 5.5 to 5.6. Several biochemical uses for Tb-3+ ion are suggested.     

RNA secondary structure as a reusable interface to biological information resources

The dissemination of biological information has become critically dependent on the Internet and World Wide Web (WWW), which enable distributed access to information in a platform independent manner. The mode of interaction between biologists and on-line information resources, however, has been mostly limited to simple interface technologies such has hypertext links, tables and forms. The introduction of platform-independent runtime environments facilitates the development of more sophisticated WWW-based user interfaces. Until recently, most such interfaces have been tightly coupled to the underlying computation engines, and not separated as reusable components. We believe that many subdisciplines of biology have intuitive and familiar graphical representations of knowledge that can serve as multipurpose user interface elements. We call such graphical idioms “domain graphics”. In order to illustrate the power of such graphics, we have built a reusable interface based on the standard two dimensional (2D) layout of RNA secondary structure. The interface can be used to represent any pre-computed layout of RNA, and takes as a parameters the sets of actions to be performed as a user interacts with the interface. It can provide to any associated application program information about the base, helix, or subsequence selected by the user. We show the versatility of this interface by using it as a special purpose interface to BLAST, Medline and the RNA MFOLD search/compute engines. These demonstrations are available at: ir|url|http://www-smi.stanford.edu/projects/helix/pubs/ gene-combis-96/     

Perylene-conjugated pyrrole polyamide as a sequence-specific fluorescent probe

Perylene-conjugated pyrrole (Py)-polyamide 2 was designed and synthesized using the Fmoc solid-phase synthesis and a subsequent Sonogashira coupling reaction with 3-bromoperylene. Interestingly, conjugate 2 did not luminesce in water at 313 nm irradiation but was turned on in the presence of target double-stranded (ds) DNA, and showed strong emission with increasing DNA concentration, in particularly, by the binding to the target telomere sequences through heterodimer formation with partner 3. Importantly, the excitation spectrum of 2 clearly indicates that the Py and Imidazole (Im) moieties in the polyamide effectively sensitize the perylene moiety to give rise to fluorescence emission. Energy transfer would occur from the Py moiety to the perylene. Thus, screening of perylene-conjugates will allow us to develop a novel "molecular light switch" with sequence-specificity.     

Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure.

Pb(2+)-catalyzed cleavage of RNA has been shown previously to be a useful probe for tertiary structure. In the present study, Pb2+ cleavage patterns were identified for ribonuclease P RNAs from three phylogenetically disparate organisms, Escherichia coli, Chromatium vinosum, Bacillus subtilis, and for E. coli RNase P RNAs that had been altered by deletions. Each of the native RNAs undergoes cleavage at several sites in the core structure that is common to all bacterial RNase P RNAs. All the cleavages occur in non-paired regions of the secondary structure models of the RNAs, in regions likely to be involved in tertiary interactions. Two cleavage sites occur at homologous positions in all the native RNAs, regardless of sequence variation, suggesting common tertiary structural features. The Pb2+ cleavage sites in four deletion mutants of E. coli RNase P RNA differed from the native pattern, indicating alterations in the tertiary structures of the mutant RNAs. This conclusion is consistent with previously characterized properties of the mutant RNAs. The Pb2+ cleavage assay is thus a useful probe to reveal alteration of tertiary structure in RNase P RNA.     

7-Aminoactinomycin as a fluorescent probe for DNA unwinding and denaturation

A fluorescent analogue of antibiotic actinomycin D, 7-aminoactinomycin D (7AAMD), which is widely used in molecular biology, was shown by steady-state, polarization, and phase fluorescent spectroscopy to bind primarily in the unwound regions of DNA with concomitant increase in its emission intensity. The maximum emission intensity of 7AAMD is observed for denatured DNA. Thus, 7AAMD may serve as a good indicator of DNA unwinding, denaturation, and fragmentation.     

Characterization of a carbocyanine derivative as a fluorescent penetration probe

The fluorescent carbocyanine dye 3,3-diheptyloxycarbocyanine [DiOC7(3)], originally described as a membrane potential probe, penetrates poorly into multicell spheroids. Since the dye is retained in the cells following spheroid disaggregation, cells can be selected from different depths within the spheroid using fluorescence-activated cell sorting. Characterization of the binding kinetics, stability, and toxicity of this probe were undertaken, and intercompared with Hoechst 33342. The optimum drug dose for achieving good separation of internal and external cells of spheroids is about tenfold lower than for Hoechst 33342, and like Hoechst, DiOC7(3) is toxic at concentrations at least tenfold higher than those required to produce a good gradient for cell separation. When cells are removed from the stain, cellular fluorescence decreases to half the initial intensity within 2 hours; however, unlike Hoechst, the carbocyanine dye does not transfer between cells.     

Equilibria in 5-S ribosomal RNA secondary structure. Bulges and interior loops in 5-S RNA secondary structure may serve as articulations for a flexible molecule

The basic assumption in this paper is that the secondary structure of a 5-S ribosomal RNA cannot be represented by a single model. We propose that the molecule can adopt, at least within the ribosome, a series of slightly different structures of nearly equal stability. The different structures arise from the existence of ambiguous base-pairing opportunities in bulged helices and the adjacent interior loops. In eubacterial 5-S RNAs there is one such an area, in eukaryotic 5-S RNAs two such areas that can give rise to structural switches. We explain how a change in secondary structure in these areas may influence the relative orientation of the surrounding helices, in other words how bulges and interior loops may serve as articulations and give rise to a flexible tertiary structure.