The 5-exonuclease domains of the DNA polymerase I proteins of Eubacteria and the FEN1 proteins of Eukarya and Archaea are members of a family of structurespecific 5-exonucleases with similar function but limited sequence similarity. Their physiological role is to remove the displaced 5 strands created by DNA polymerase during displacement synthesis, thereby creating a substrate for DNA ligase. In this paper, we define the substrate requirements for the 5-exonuclease enzymes from Thermus aquaticus, Thermus thermophilus, Archaeoglobus fulgidus, Pyrococcus furiosus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum. The optimal substrate of these enzymes resembles DNA undergoing strand displacement synthesis and consists of a bifurcated downstream duplex with a directly abutted upstream duplex that overlaps the downstream duplex by one base pair. That single base of overlap causes the enzymes to leave a nick after cleavage and to cleave several orders of magnitude faster than a substrate that lacks overlap. The downstream duplex needs to be 10 base pairs long or greater for most of the enzymes to cut efficiently. The upstream duplex needs to be only 2 or 3 base pairs long for most enzymes, and there appears to be interaction with the last base of the primer strand. Overall, the enzymes display very similar substrate specificities, despite their limited level of sequence similarity.The 5Ј nuclease domains of DNA polymerase I from Escherichia coli and Thermus aquaticus were the first extensively characterized members of a large class of structure-specific 5Ј-exonucleases (1, 2). Initially it was proposed that these enzymes work as true exonucleases removing predominantly mono-or dinucleotides from the 5Ј end of double-stranded DNA (3). More detailed studies have shown that 5Ј nucleases of this type specifically recognize bifurcated ends of double-stranded regions and remove single-stranded 5Ј arms by cutting the phosphodiester bond after the first base pair of the duplex, leaving a 3Ј hydroxyl end (2). A mammalian enzyme with functional similarity to the 5Ј-exonuclease domain of E. coli polymerase I was isolated nearly 30 years ago (4). Later, additional members of this group of enzymes called flap endonucleases (FEN1) from Eukarya and Archaea were shown to possess a nearly identical structure-specific activity (5-8), although they have limited sequence similarity to the bacterial 5Ј-exonuclease proteins.The substrate specificities of the FEN1 enzymes and the eubacterial and related bacteriophage enzymes have been examined and found to be similar for all enzymes (2, 5, 6, 8 -11). The minimal requirement for cleavage is a bifurcated duplex with a free 5Ј end. The presence of an upstream primer that directly abuts the downstream strand stimulates cleavage, but its precise effect on the site of cleavage remains unclear. In the majority of studies that were done with the flap substrate described in Harrington et al. (5), the enzymes leave predominately a 1-nucleotide gap or 1-nucleotide overlap between t...

The short lengths of microRNAs (miRNAs) present a significant challenge for detection and quantitation using conventional methods for RNA analysis. To address this problem, we developed a quantitative, sensitive, and rapid miRNA assay based on our previously described messenger RNA Invader assay. This assay was used successfully in the analysis of several miRNAs, using as little as 50-100 ng of total cellular RNA or as few as 1,000 lysed cells. Its specificity allowed for discrimination between miRNAs differing by a single nucleotide, and between precursor and mature miRNAs. The Invader miRNA assay, which can be performed in unfractionated detergent lysates, uses fluorescence detection in microtiter plates and requires only 2-3 h incubation time, allowing for parallel analysis of multiple samples in high-throughput screening analyses.

The invasive signal amplification reaction is a sensitive method for single nucleotide polymorphism detection and quantitative determination of viral load and gene expression. The method requires the adjacent binding of upstream and downstream oligonucleotides to a target nucleic acid (either DNA or RNA) to form a specific substrate for the structure-specific 5' nucleases that cleave the downstream oligonucleotide to generate signal. By running the reaction at an elevated temperature, the downstream oligonucleotide cycles on and off the target leading to multiple cleavage events per target molecule without temperature cycling. We have examined the performanceof the FEN1 enzymes from Archaeoglobus fulgidus and Methanococcus jannaschii and the DNA polymerase I homologues from Thermus aquaticus and Thermus thermophilus in the invasive signal amplification reaction. We find that the reaction has a distinct temperature optimum which increases with increasing length of the downstream oligonucleotide. Raising the concentration of either the downstream oligonucleotide or the enzyme increases the reaction rate. When the reaction is configured to cycle the upstream instead of the downstream oligonucleotide, only the FEN1 enzymes can support a high level of cleavage. To investigate the origin of the background signal generated during the invasive reaction, the cleavage rates for several nonspecific substrates that arise during the course of a reaction were measured and compared with the rate of the specific reaction. We find that the different 5' nuclease enzymes display a much greater variability in cleavage rates on the nonspecific substrates than on the specific substrate. The experimental data are compared with a theoretical model of the invasive signal amplification reaction.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.