Substrate ( Solid Phase Peptide Synthesis ) a OH TGf , , - 6 O 7 o N O HI NH 2 Linker + TG - Substrate

A reaction cassette has been designed for the highly sensitive detection of the making and breaking of chemical bonds. The system is envisioned as a companion device to be used in the search for antibody and other novel catalysts. The cassette also may have important clinical applications in the design of diagnostic reagents. In its fully encoded format, this methodology is capable of both detecting and decoding chemical events. The essence of antibody catalysis is the creation of a library of molecules from which those with unique chemical potential are selected. As for any application of combinatorial chemistry, the successful identification of useful molecules depends not only on the chemical insight that was used to create the library but also on the ease and sensitivity by which the library can be screened (1). In essence, the problem reduces to the separate issues of display and detection. In the case of antibodies, the conversion of diversity into combinatorial libraries in phage where the recognition and replication functions are linked in a single entity has allowed simple binding events to be monitored, thus solving the display problem. However, the problem of the direct detection of antibody catalysts at the level of phage particles remains (2, 3). To accomplish this, one needs to detect catalysis at the level of only a few molecules (4, 5) either by use of selection or highly sensitive assays (6). Here, we report success with a concept wherein a reaction cassette is constructed that should be capable of detecting the making or breaking of any chemical bond, even when only a few molecules of product are formed. In principle, the cassette can be considered to be a companion assay device that can be constructed for any system in which a new catalyst or reagent is sought. MATERIALS AND METHODS Substrate Synthesis. The peptide was assembled manually on TentaGel support (Novabiochem or Rapp Polymere) according to standard fluorenylmethoxycarbonyl (Fmoc) methodology (7). To have a final loading of 40-60 ,umol of peptide per g of support, the first step of the synthesis was performed with a 4-fold excess of solid support to. Fmoc amino acids; after all final washings, the unreacted amino groups were capped by treating twice for 10 min each with 0.25 volume of 4.23 M acetic anhydride in 2,6-lutidine/0.75 volume of 0.53 M N,Ndimethylaminopyridine in tetrahydrofuran (THF). The overall yield for peptide synthesis was -98%. The tert-butyl (t-Bu) protecting group for the hydroxyl moiety on tyrosine was removed by treatment with 5% (vol/ vol) ethanedithiol in anhydrous CF3COOH for 2 h, followed by extensive washing with dichloromethane (DCM), methanol, and N,N-dimethylformamide. The Fmoc protecting group was removed before coupling to the linker L-I (4-(O-dimethoxytrityl)hydroxybutyrate, sodium salt; see Fig. 3). The matrix containing tyrosine-O-L-I was converted to tyrosine-O-acetyl after selective deprotection of the phenolic ring (by treatment with concentrated NH40H for 3 h) and capping (treatment with 0.25 volume of 4.23 M acetic anhydride, in 2,6-lutidine/ 0.75 volume of 0.53 M N,N-dimethylaminopyridine in THF for 30 min). The yield after each step was determined by the dimethoxytrityl cation assay (8). Linker L-I was prepared in one step from the sodium salt of 4-hydroxybutyrate and dimethoxytrityl chloride in pyridine (9). DNA Synthesis. DNA synthesis was carried out on a 394 Applied Biosystems DNA synthesizer using standard phosphoramidite chemistry (10). The standard 1 Amol cycle was modified as follows: (i) Washing steps 3, 59, 61, 66, 77, and 94 were prolonged to 30 s (the use of longer or shorter times decreased the yield); (ii) the incubation time with phosphoramidite and tetrazole (step 45) was prolonged from 25 s to 120 s; and (iii) the concentration of the phosphoramidites was increased from 0.1 M to 0.2 M. Under these conditions the average yield per step was -97%. The bases were deprotected upon treatment with concentrated NH40H for 20 h at 55°C. The dimethoxytrityl group is removed upon treatment with 3% CCl3CO2H in DCM for 5 min, followed by extensive washing with DCM, THF, methanol, 20 mM Tris HCl, pH 8/160 mM NaCl, and distilled water (dH2O). After this step, the cassette is ready for use. Enzymatic Cleavage and Inhibition Experiments. The cassette (1 mg; 5.9 p,mol of substrate peptide-polynucleotide hybrid per g of solid support) was suspended in 20 ,ul of 20mM Tris HCl, pH 8/160 mM NaCl and 170 ,u of dH2O; 0.85 nmol of trypsin, pepsin, papain, carboxypeptidase A, ca-chymotrypsin, or a-chymotrypsin and 1 mg of Bowman-Birk inhibitor (11) in 10 ,ul of dH20 was added to the reaction medium, and the mixture was shaken at 20°C. Supernatant fluids (18.7 ,ul) were taken after 30 min and were submitted to the PCR. PCR Experiments. Aliquots (18.7 ,ul) from the reaction mixture were mixed with the following PCR components (Promega): 1.2 ,tl of 2.5 mM MgCl2, 2 ,ul of Taq buffer, 1.6 ,l of 2.5 mM deoxynucleotide triphosphates, and 1 ,ul of primers at 100 pmol/,l. Taq polymerase (2.5 units; 0.5 ,ul) was added just before starting the first PCR cycle. A positive control (PCR components only) was run with dH2O containing 1 pmol of the polynucleotide sequence used in this study. A negative control was run under the same conditions without the polynucleotide sequence. The PCR was run on a Perkin-Elmer/ Cetus 9600 instrument with the following cycle program: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. After 35 cycles the results were analyzed on agarose gels [1% GIBCO/BRL agarose/2% FMC NuSieve GTG agarose with 90 mM Tris/64.6 mM borate/2.5 mM EDTA, pH 8.3 (lx TBE) at 103 mV]. Fluorescence Assay. After the PCR, the reaction medium (25 ,ul) was transferred to a 96-well ELISA plate and diluted Abbreviations: Fmoc, fluorenylmethoxycarbonyl; DCM, dichloromethane; THF, tetrahydrofuran; dH20, distilled water; t-Bu, tertbutyl. *To whom reprint requests should be addressed. 2278 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad Sci. USA 92 (1995) 2279 PCR on Solid Support