Fluorescence Assay Platform Design
The common property of all sequence-specific DNA-binding proteins is their ability to bind with high affinity and specificity to a DNA duplex containing a unique nucleotide sequence, i.e., the DNA-binding site for the protein. Mediomics’ novel assay platform relies on this common characteristic. A DNA duplex containing the sequence-specific DNA-binding site for a given target protein is split into two DNA “half-site” duplexes each having a short single-stranded overhang. These single-stranded extensions are short enough so that in the absence of the target protein little spontaneous re-association occurs. When the target protein is present, however, its high affinity for the full-length DNA sequence will drive the re-association of the two half-site DNA duplexes. This re-association can be sensitively detected by incorporating an appropriate fluorescence probe into each one of the two DNA half-sites. The presence of the DNA-binding protein is detected as a change in fluorescence signal. A simple variation of this basic platform design allows a DNA-binding protein to function as a sensitive biosensor for its specific small molecule co-regulator (ligand) as represented schematically below:

L-Methionine (L-Met)
L-Methionine is an essential amino acid that must be obtained from food sources or from dietary supplements. It is a precursor for the other sulfur amino acids, cystine, taurine, and glutathione. Methionine reacts with adenosine triphosphate to form S-adenosyl methionine (SAM), which is the principal methyl donor in the body and contributes to the synthesis of many important substances, including epinephrine and choline. L-methionine is also an important supplement for animal feed. Currently, L-methionine is quantified using the high pressure liquid chromatography (HPLC) method. HPLC is time consuming, costly and, due to the large amount of organic solvent required, not environmentally friendly.

Bridge-It^® L-Methionine Fluorescence (L-Met) Assay^1,2

Eukaryotic cells contain an estimated 3,000 sequence-specific DNA binding proteins. These important proteins, acting either with or without a specific small molecule coregulator (ligand), control all aspects of genomic DNA activity including gene expression, DNA replication, and DNA repair. Mediomics is applying its novel fluorescence assay platform to develop in vitro assays useful for rapidly and sensitively quantifying the activity of both DNA binding proteins and their small molecule ligands.

The Mediomics Bridge-It^® L-methionine fluorescence assay method is based on a combination of well-established fluorescence measurement techniques and a new assay platform design that utilizes DNA-binding proteins as biosensors for their respective small molecule coregulators (ligands). The affinity of the DNA sequence-specific MetJ methionine repressor protein for its unique DNA binding site is greatly increased in the presence of its ligand, S-adenosyl methionine (SAM). For this assay, the MetJ consensus sequence was split into two approximately equal DNA “half-sites” with one half fragment labeled with fluorescein and the other half fragment labeled with Oyster^® 645 fluorophore^3,4. The relative amount of SAM present in a test sample will influence the amount of DNA-MetJ protein complex formation in the assay. When this complex forms, it brings the fluorescence labeled-DNA half-sites into close proximity and causes a measurable change (increase) in fluorescence signal emission that can be readily measured using a microplate reader (wavelength settings: absorption 485 nm; emission 665 nm). The L-methionine is readily converted to SAM by S-adenosylmethionine synthetase in the presence of ATP and Mg²⁺. L-Methionine concentrations in test samples are determined using an L- methionine standard curve.

The Bridge-It^® L-methionine fluorescence assay method exhibits highly desirable performance characteristics including a high signal to background (S/B) ratio, a good linear dynamic range, and, a detection sensitivity of ~5 μM. The total assay time is 45 minutes.

In contrast to the HPLC procedure, the Bridge-It^® L-Methionine fluorescence assay method utilizes the 384 and 96-well microplate format. Thus, it is ideally suited for the rapid, simultaneous measurement of L-methionine levels in large numbers of test samples. Also, this method is readily adaptable to the high-throughput screening platforms currently being used in drug discovery research. In comparison with the HPLC procedure, the Bridge-It^® L-methionine fluorescence assay method is:

Easy
Mix test sample or standard with the Assay solution and incubate
Fast
Read fluorescent signal after 45 minutes of incubation
Sensitive
Assay measures L-Mehionine at a lower limit of sensitivity of 5 µM
Flexible
Assay is adaptable to both low- and high-throughput screening formats

Additional information on the Bridge-It^® L-methionine fluorescence assay protocol and product ordering information are available on-line at www.mediomics.com. This product is protected by US Patent 6,544,746.