Electrophoretic selection with capillary electrophoresis (CE) is used, for the first time, to isolate functional nucleic acid sequences using SELEX (systematic evolution of ligands by exponential enrichment). SELEX uses molecular evolution to select functional sequences (aptamers) from random RNA or DNA libraries. Conventional SELEX is usually performed with affinity chromatography, which may introduce significant bias into the selection step. Important biases include the slow kinetics involved in the elution of strongly bound sequences and performing the selection with the target molecule tethered to the stationary support, not in free solution. In this novel CE-SELEX approach, selection occurs in free solution. The nucleic acid sequences that bind the target undergo a mobility shift, migrating at a different rate, allowing them to be separated from the inactive sequences. Thus, there is no need to wash the active sequences off a column as in conventional SELEX, eliminating any kinetic bias. In this work, the viability of CE-SELEX was demonstrated by performing selections against immunoglobulin E (IgE). Anti-IgE aptamers with dissociation constants as low as 40 nM were obtained in only two rounds of selection.
We have engineered aptamers that contain fluorescent reporters and that signal the presence of cognate ligands in solution. Two different anti-adenosine "signaling aptamers", one made from RNA and one from DNA, can selectively signal the presence of adenosine in solution. Increases in fluorescence intensity reproducibly follow increases in adenosine concentration, and can be used for quantitation. The facile methods we have developed can potentially be used for generating a wide variety of signaling aptamers for use in sensor arrays.
Aptamers with high affinity for IgE were selected using capillary electrophoresis to demonstrate the compatibility of this technique with SELEX. The high selectivity and efficiency of CE gave rise to a very high rate of enrichment, allowing high-affinity, high-selectivity aptamers to be obtained in only four rounds of selection. Decreasing the number of rounds shortens the selection procedure from the 4-6 weeks typical of SELEX to several days. The use of "bulk" dissociation constant measurements was introduced as a method for assessing the DNA pool after each round of selection. The average dissociation constant of the sequences in the DNA pool for IgE after four rounds of selection was 29 nM. The distribution of the dissociation constants for the sequences in the pool was very narrow with a standard deviation of only 6 nM. All of the sequences assessed exhibited high specificity for human IgE when compared with human IgG or mouse IgE.
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