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COMMUNICATIONS otides in length. In vitro "evolution" was done on this region at 30% mutagenesis. and four more rounds of in vitro selection followed before this second population was cloned. From these sequences, a consensus region was discovered. Certainly though this work is a pioneering achievement in the field, it is an example of how the conventional protocol is significantly more involved than that presented here.In summary, a novel in vitro selection protocol has been designed to take advantage of a combinatorial library of small size that has multiple copies of every distinct sequence. The method condensed the many days of a typical screening strategy to less than two days. This was a proof-of-concept experiment that showed that the new method succeeds by creating a large number of copies of individual sequences in the initial random pool, consistently reducing the level of nonspecific binding sequences per selection round, and effectively amplifying the few surviving sequences.Since only the original synthesized sequences were used for all the screenings, the technique should allow for the iterative in vitro selection of modified oligonucleotides that previously could not undergo this powerful process."01 Hence, this method should significantly increase the power of the in vitro selection method and is the direction that we are currently investigating. Experimental SectionThe DNA library[4] (31 mg) was labeled at the Yend with [y-"P]ATP, purified by gel chromatography, and suspended in 300 mL of folding buffer (300 mM KCI, 5 mM MgCI,. 20mM Tris. pH 7 5 ) . After cooling down to room temperature following denaturation at 75 C, the "P-labeled DNA was loaded onto an acetate-agarose precolumn (300 pL), which was attached directly to a 2.5mM ATP-agarose column (800 pL, Sigma). The precolumn was washed with 600 pL of buffer, and the eluted DNA was allowed to equilibrate on the ATP-agarose column for 10 min. The precolumn was discarded after a single use as were all subsequent columns. After equilibration, the ATP-agarose column was washed with 4 mL of folding buffer to elute unbound or weakly bound oligonucleotides. The retained DNA was eluted with 3 mL of the ATP elution buffer (5mM ATP in folding buffer) and collected in 500 pL fractions.In order to perform another round ofselection, the ATP had to be removed. Hence, the eluted fractions were collected directly into Microcon-3 microcentrifuge devices (3000 D cutoff, Amicon) After membrane diafiltration. about 98% of the total ATP was removed. The filtered fractions were then pooled, and folding buffer was added until a final volume of 10 mL was attained. Theconcentration ofcontaminating ATP concentration was 3 0 p~ for the DNA sample, which was over 80 times more dilute than that of the 2 . 5 m~ ATP-agarose column. Each cycle of selection started with a new set of stacked affinity columns, i.e. a precolumn attached to a ligand column. The screening cycles for the ATP aptamers are summarized in Table 1 The rare-DNA PCR was performed as follows: On the last c...

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