No abstract

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10 7 reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.

Structures of a number of ribosomal proteins have now been determined by crystallography and NMR, though the complete structure of a ribosomal protein-rRNA complex has yet to be solved. However, some ribosomal protein structures show strong similarity to well-known families of DNA or RNA binding proteins for which structures in complex with cognate nucleic acids are available. Comparison of the known nucleic acid binding mechanisms of these non-ribosomal proteins with the most highly conserved surfaces of similar ribosomal proteins suggests ways in which the ribosomal proteins may be binding RNA. Three binding motifs, found in four ribosomal proteins so far, are considered here: homeodomain-like alpha-helical proteins (L11), OB fold proteins (S1 and S17) and RNP consensus proteins (S6). These comparisons suggest that ribosomal proteins combine a small number of fundamental strategies to develop highly specific RNA recognition sites.

Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H 2 0 and D 2 0 solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 2 0.5 and 4.6 ? 0.5 water molecules coordinated to Eu3+ bound to the nl site of unadenylylated enzyme, GSo, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 ? 0.5 water molecules coordinated to Eu3+ at site n2 for both GSo and GSl2. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 ? 1.5 A. Distances between a Tb" ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site nl was observed to decrease from Kd = 0.35 2 0.09 pM for GSo to Kd = 0.06 ? 0.02 pM for GSI2. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 2 0.9 pM and Kd = 2.6 2 0.7 pM for GSo and GSI2, respectively. Competition experiments indicate that Mn2+ affinity at site nl decreases as a function of increasing adenylylation state, from Kd = 0.05 2 0.02 pM for GSo to K d = 0.35 ? 0.09 pM for GSI2. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 ? 1.3 pM for GSo and Kd = 4.0 ? 1.0 pM for GSI2). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991. Biochemistry 30: 1413-141 8), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities.