Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy.

Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule detection (SMD) techniques and related approaches, such as fluorescence correlation spectroscopy (FCS), stochastically sample the fluorescence properties of individual constituent molecules and only then average many such detection events to define the properties of the assay system as a whole. Analysis of single molecular events is accomplished using confocal optics with an illumination/detection volume of approximately 1 fl (10(-15) L) such that the signal is insensitive to miniaturization of HTS assays to 1 µl or below. In this report we demonstrate the general applicability of one SMD technique (FCS) to assay configuration for target classes typically encountered in HTS and confirm the equivalence of the rate/equilibrium constants determined by FCS and by macroscopic techniques. Advantages and limitations of the current FCS technology, as applied here, and potential solutions, particularly involving alternative SMD detection techniques, are also discussed.

Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs.

In several forms of beta-thalassemia, mutations in the second intron of the beta-globin gene create aberrant 5' splice sites and activate a common cryptic 3' splice site upstream. As a result, the thalassemic beta-globin pre-mRNAs are spliced almost exclusively via the aberrant splice sites leading to a deficiency of correctly spliced beta-globin mRNA and, consequently, beta-globin. We have designed a series of vectors that express modified U7 snRNAs containing sequences antisense to either the aberrant 5' or 3' splice sites in the IVS2-705 thalassemic pre-mRNA. Transient expression of modified U7 snRNAs in a HeLa cell line stably expressing the IVS2-705 beta-globin gene restored up to 65% of correct splicing in a sequence-specific and dose-dependent manner. Cell lines that stably coexpressed IVS2-705 pre-mRNA and appropriately modified U7 snRNA exhibited up to 55% of permanent restoration of correct splicing and expression of full-length beta-globin protein. This novel approach provides a potential alternative to gene replacement therapies.

Analysis of rate-determining conformational changes during self-splicing of the Tetrahymena intron.

RNA catalyzed reactions are often limited in vitro by the rate of structural rearrangements in the RNA. Analysis of intra- and intermolecular splicing of the Tetrahymena preribosomal RNA revealed two well resolved kinetic phases with rate constants of approximately 2.5 and 0.02 min-1 at 30 degrees C. The data are consistent with a model in which the second phase results from slow refolding of the pre-rRNA. Point mutations result in redistribution of the RNA among different conformations that can be detected by native gel electrophoresis. The active pre-rRNA rapidly progresses to a product complex in the presence of GTP. Release of the ligated exons is slightly slower than splicing at 30 degrees C (0.3 -0.5 min-1). In contrast, the intermediate complex after the first step of splicing dissociates much more slowly (5 x 10(-3) min-1), accounting for the low amount of intron-3' exon intermediate typically seen during splicing of wild type pre-rRNA. These results provide an initial framework for studying conformational changes that accompany excision of the Tetrahymena intron from ribosomal RNA.

Fingerprinting the folding of a group I precursor RNA.

Evidence that folding of the Tetrahymena pre-rRNA follows a defined path and is rate-determining for splicing at physiological temperatures is presented. Structural isomers were separated by native polyacrylamide gel electrophoresis and their splicing activities were compared. GTP binding selectively shifts the active form of the pre-RNA to an electrophoretic band containing both spliced and unspliced RNA. In situ chemical modification provides evidence for base-pair rearrangements in the 5' exon and structural alterations in the intron core of partially and fully active forms. Transition to the fully active precursor requires high temperature, but the activation energy is lower than expected for opening of RNA helices. Implications for control of RNA conformation during splicing are discussed.

Self-splicing of the Tetrahymena pre-rRNA is decreased by misfolding during transcription.

RNA processing depends in part on the ability of nascent transcripts to fold into the desired conformation. Self-splicing of the group I intron from Tetrahymena was used to assess the folded state of preribosomal RNA transcripts when synthesized in vitro. A simple method for isolating nondenatured RNA from a T7 RNA polymerase reaction was tested. The intron alone is fully active when transcribed at 30 degrees C, suggesting that the active structure is both kinetically and thermodynamically favored. Longer precursor RNAs, however, were less than completely active in self-splicing. Full activity, as judged by both the initial rate and the extent of product formation, was restored by brief incubation at 95 degrees C and rapid cooling in the presence of magnesium ion. This result did not depend on the length of the precursor RNA in any simple way, but correlated loosely with the presence of intact exon domains. When transcribed in the absence of cellular proteins, a significant portion of the pre-RNA appears to be trapped in a conformation that does not readily undergo the first step of splicing.

Substitution of glutamine for glutamic acid-58 in Escherichia coli thymidylate synthase results in pronounced decreases in catalytic activity and ligand binding.

The recent determination of the crystal structure of Escherichia coli thymidylate synthase (TS) [Matthews et al. (1989) J. Mol. Biol. 205, 449-454] has implicated the glutamic acid residue at position 58 in a mechanistic role which could involve the interaction of its gamma-carboxyl side chain with the nucleotide substrate and/or the folate cofactor. The site-specific mutagenesis of Glu-58 to Gln-58 in E. coli TS provided the opportunity to explore its functional role in activity and binding. When profiled by the spectrophotometric and tritium release assays, the 370- and 760-fold decreases, respectively, in kcat and the elevated Km values for the Gln-58 mutant enzyme indicated a significant involvement of Glu-58 in substrate binding and turnover. The apparent dissociation constant for the covalent FdUMP-enzyme binary complex was 30 microM, which is 5-fold higher than that found for the wild-type enzyme, while the inhibitory ternary complex apparent dissociation constants for FdUMP and CH2H4folate for the Gln-58 enzyme were 10- and 60-fold higher, respectively, than those for the wild-type enzyme under saturating conditions. The extent of covalent FdUMP binding to the Gln-58 enzyme was reduced from 1.5 to 0.7 per dimer in the inhibitory ternary complex but only from 0.7 to 0.5 per dimer in the binary complex of the Gln-58 enzyme. The usual 2.1-fold enhancement of FdUMP binding to wild-type TS in the presence of CH2H4folate was not observed for the Gln-58 enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

An alternative helix in the 26S rRNA promotes excision and integration of the Tetrahymena intervening sequence.

A highly conserved ribosomal stem-loop immediately upstream of the Tetrahymena splice junction can inhibit both forward and reverse self-splicing by competing with base pairing between the 5' exon and the guide sequence of the intervening sequence. Formation of this unproductive hairpin is preferred in precursor RNAs with short exons and results in a lower rate of splicing. Inhibition of self-splicing is not observed in longer precursors, suggesting that additional interactions in the extended exons can influence the equilibrium between the productive and unproductive hairpins at the 5' splice site. An alternative pairing upstream of the 5' splice site has been identified and is proposed to stabilize the active conformer of the pre-rRNA. Nucleotide changes that alter the ability to form this additional helix were made, and the self-splicing rates were compared. Precursors in which the proposed stem is stabilized splice more rapidly than the wild type, whereas RNAs that contain a base mismatch splice more slowly. The ability of DNA oligomers to bind the RNA, as detected by RNase H digestion, correlates with the predicted secondary structure of the RNA. We also show that a 236-nucleotide RNA containing the natural splice junction is a substrate for intervening sequence integration. As in the forward reaction, reverse splicing is enhanced in ligated exon substrates in which the alternative rRNA pairing is more stable.