Fast high-resolution mapping of long fragments of genomic DNA based on single-molecule detection.

Here we describe bacterial genotyping by direct linear analysis (DLA) single-molecule mapping. DLA involves preparation of restriction digest of genomic DNA labeled with a sequence-specific fluorescent probe and stained nonspecifically with intercalator. These restriction fragments are stretched one by one in a microfluidic device, and the distribution of probes on the fragments is determined by single-molecule measurement of probe fluorescence. Fluorescence of the DNA-bound intercalator provides information on the molecule length. Because the probes recognize short sequences, they encounter multiple cognate sites on 100- to 300-kb-long DNA fragments. The DLA maps are based on underlying DNA sequences of microorganisms; therefore, the maps are unique for each fragment. This allows fragments of similar lengths that cannot be resolved by standard DNA sizing techniques to be readily distinguished. DNA preparation, data collection, and analysis can be carried out in as little as 5h when working with monocultures. We demonstrate the ability to discriminate between two pathogenic Escherichia coli strains, O157:H7 Sakai and uropathogenic 536, and we use DLA mapping to identify microorganisms in mixtures. We also introduce a second color probe to double the information used to distinguish molecules and increase the length range of mapped fragments.

Probing the dynamics of the P1 helix within the Tetrahymena group I intron.

RNA conformational transformations are integral to RNA's biological functions. Further, structured RNA molecules exist as a series of dynamic intermediates in the course of folding or complexation with proteins. Thus, an understanding of RNA folding and function will require deep and incisive understanding of its dynamic behavior. However, existing tools to investigate RNA dynamics are limited. Here, we introduce a powerful fluorescence polarization anisotropy approach that utilizes a rare base analogue that retains substantial fluorescence when incorporated into helices. We show that 6-methylisoxanthopterin (6-MI) can be used to follow the nanosecond dynamics of individual helices. We then use 6-MI to probe the dynamics of an individual helix, referred to as P1, within the 400nt Tetrahymena group I ribozyme. Comparisons of the dynamics of the P1 helix in wild type and mutant ribozymes and in model constructs reveal a highly immobilized docked state of the P1 helix, as expected, and a relatively mobile "open complex" or undocked state. This latter result rules out a model in which slow docking of the P1 helix into its cognate tertiary interactions arises from a stable alternatively docked conformer. The results are consistent with a model in which stacking and tertiary interactions of the A(3) tether connecting the P1 helix to the body of the ribozyme limit P1 mobility and slow its docking, and this model is supported by cross-linking results. The ability to isolate the nanosecond motions of individual helices within complex RNAs and RNA/protein complexes will be valuable in distinguishing between functional models and in discerning the fundamental behavior of important biological species.

An automated sample preparation system with mini-reactor to isolate and process submegabase fragments of bacterial DNA.

Existing methods for extraction and processing of large fragments of bacterial genomic DNA are manual, time-consuming, and prone to variability in DNA quality and recovery. To solve these problems, we have designed and built an automated fluidic system with a mini-reactor. Balancing flows through and tangential to the ultrafiltration membrane in the reactor, cells and then released DNA can be immobilized and subjected to a series of consecutive processing steps. The steps may include enzymatic reactions, tag hybridization, buffer exchange, and selective removal of cell debris and by-products of the reactions. The system can produce long DNA fragments (up to 0.5 Mb) of bacterial genome restriction digest and perform DNA tagging with fluorescent sequence-specific probes. The DNA obtained is of high purity and floating free in solution, and it can be directly analyzed by pulsed-field gel electrophoresis (PFGE) or used in applications requiring submegabase DNA fragments. PFGE-ready samples of DNA restriction digests can be produced in as little as 2.1 h and require less than 10(8) cells. All fluidic operations are automated except for the injection of the sample and reagents.

Single-molecule fluorescence of nucleic acids.

Less than a decade old, single-molecule fluorescence of nucleic acids has rapidly become an important tool in the arsenal of biological probes. A variety of novel approaches to investigate conformational dynamics, catalytic mechanisms, folding pathways and protein-nucleic-acid interactions have recently been devised for nucleic acids using this technique. Combined with biomechanical tools and ensemble measurements, single-molecule fluorescence methods extend our ability to observe and understand biomolecules and complex biological processes.

The global conformation of the hammerhead ribozyme determined using residual dipolar couplings.

The global structure of the hammerhead ribozyme was determined in the absence of Mg(2+) by solution NMR experiments. The hammerhead ribozyme motif forms a branched structure consisting of three helical stems connected to a catalytic core. The (1)H-(15)N and (1)H-(13)C residual dipolar couplings were measured in a set of differentially (15)N/(13)C-labeled ribozymes complexed with an unlabeled noncleavable substrate. The residual dipolar couplings provide orientation information on both the local and the global structure of the molecule. Analysis of the residual dipolar couplings demonstrated that the local structure of the three helical stems in solution is well modeled by an A-form conformation. However, the global structure of the hammerhead in solution in the absence of Mg(2+) is not consistent with the Y-shaped conformation observed in crystal structures of the hammerhead. The residual dipolar couplings for the helical stems were combined with standard NOE and J coupling constant NMR data from the catalytic core. The NOE data show formation of sheared G-A base pairs in domain 2. These NMR data were used to determine the global orientation of the three helical stems in the hammerhead. The hammerhead forms a rather extended structure under these conditions with a large angle between stems I and II ( approximately 153 degrees ), a smaller angle between stems II and III ( approximately 100 degrees ), and the smallest angle between stems I and III ( approximately 77 degrees ). The residual dipolar coupling data also contain information on the dynamics of the molecule and were used here to provide qualitative information on the flexibility of the helical domains in the hammerhead ribozyme-substrate complex.