Bridging Genomics and Proteomics

Image: Courtesy of Aclara Biosciences The eTag™ system from ACLARA BioSciences of Mountain View, Calif., enables the multiplexed, solution-phase analysis of proteins and nucleic acids. This technology employs the company's eTag reporters, which are low-molecular weight, fluorescent molecules that are readily separated and quantified by capillary electrophoresis (CE) in conjunction with fluorescence detection using standard capillary DNA sequencers. This method can be used for any application to simultaneously quantify multiple protein or nucleic acid targets. Its potential for multiplexing will make it attractive to researchers performing proteomic and genomic analyses, including those in drug discovery and development. "Finding candidate therapeutic targets and potential structural leads no longer represents major hurdles in drug discovery. Today's challenge is to select the most promising targets and leads from an already extensive and rapidly growing number of alternatives," comments Jon Miller, ACLARA's director of corporate development. "To improve the quality of their selections, researchers are using patterns of gene and protein expression that reflect health or disease, as well as therapeutic and toxic effects. Implementing this 'systems biology' approach requires analysis of expression patterns of multiple genes and proteins in large numbers of samples." ACLARA has developed about 400 different eTag reporters, and is working to further broaden that repertoire. The first set of eTag reporters employed chemical derivatives of fluorescein, and the company has also developed eTag-reporter libraries that feature fluorophores emitting other, spectrally distinct colors. Researchers can achieve increased levels of multiplexing by combining differently colored reporters. All eTag assays are conducted in a completely solution-phase, mix-and-read format that uses cell lysates without prepurification of RNA or protein. Each assay employs a target-recognition element attached to a different eTag reporter via a cleavable linkage. The assay components are mixed and incubated so that the eTag-containing reagents can bind to their respective targets. Next, a selective cleavage step is performed. Unbound eTag-containing reagents remain intact. In contrast, the linkages tethering the eTag reporters to the target-bound reagents are specifically cleaved, releasing the eTags for subsequent separation and quantification. The identity of each released fluorescent eTag reporter reveals that of the recognition element to which it was originally attached. It is not necessary to remove eTag reagents that did not bind to their respective targets, because any such unused reagents are separated out during the electrophoretic analysis, along with all other assay components. For protein assays, each eTag reporter is linked chemically to a specific antibody or other protein-recognition element. After binding of the eTag-labeled antibody to its target protein, that linkage is cleaved in target-bound, but not free, conjugates by means of a radical-mediated reaction. ACLARA has developed several assay formats for multiplex protein assays, including "sandwich" assays employing two antibodies, with the chemical cleavage agent on the second antibody; competitive assays, with the cleavage agent on the competing antigen; and assays for detecting cell- surface antigens or ligand-receptor binding, with the cleavage agent fixed to the cell membrane via a lipid moiety. For mRNA assays, ACLARA employs a simplified version of the Invader® assay from Third Wave Technologies of Madison, Wis.1 Briefly, two target-specific oligonucleotides are employed: the signal oligonucleotide, containing a 5'-noncomplementary arm; and a second, partially overlapping Invader oligonucleotide. The resulting ternary complex is recognized specifically by a structure- specific endonuclease (Third Wave's Cleavase® enzyme), which cleaves the 5' arm from target-bound, but not free, signal oligonucleotide. In ACLARA's adaptation, eTag reporters are linked chemically to the 5'-noncomplementary arm, and are released for separation and quantification by CE after just one cleavage reaction. Like the standard Invader assay, the eTag multiplex Invader assays are isothermal--no thermal cycler is required--with incubations at 65°C, the melting temperature of the signal oligonucleotides. This elevated temperature would denature most proteins, precluding the execution of multiplexed mRNA and protein assays in the same reaction vessel. However, the ultimate analysis step is identical. Researchers measuring both mRNAs and proteins in the same sample simply divide the cell lysate into two aliquots, perform the two multiplexed assays using different sets of eTag reporters, and then combine the two assay mixtures prior to CE analysis. ACLARA has assembled predeveloped panels of eTag-multiplexed assays and will also develop custom assay panels based on client specifications, along with all requisite assay reagents and relevant standards. The separately purchased eTag Informer™ software identifies and quantifies each eTag reporter, and calculates the amount or concentration of each analyte. --Deborah A. Fitzgerald 1. D. A. Wilkinson, "Invasive action," The Scientist, 13[22]:16, Nov. 8, 1999.

Bridging Genomics and Proteomics

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