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ANALYTICAL CURRENTS Improved SELEX
again. As many as 18 rounds of 75 SELEX may be required. Bowser and Mendonsa’s new CE-SELEX technique, however, 50 does not involve columns. Binding occurs in solution, which 25 frees up the entire target molecule for interactions. Bound se0 quences undergo a mobility shift Initial Round 1 Round 2 Round 3 Round 4 in CE, which facilitates their separation from unbound sequences. Because washing steps are Percentage of isolated DNA aptamers that bind IgE unnecessary, researchers can isoafter each CE-SELEX round. late high-affinity aptamers. human IgG or mouse IgE. In contrast, The researchers demonstrate CEonly ~50% binding is typically observed SELEX with human IgE as the target after the first few rounds of SELEX. molecule, which was chosen because it In addition to improving enrichment, has been well characterized by conventhe CE-SELEX method is much faster tional SELEX. A DNA library was used than its conventional counterpart. Bowsfor aptamer identification. Only four rounds of CE-SELEX were necessary. In er and Mendonsa isolated aptamers withfact, nearly 100% of the DNA sequences in 2– 4 days using CE-SELEX, whereas typical versions of the technique can take isolated after two rounds showed affinias long as a month to yield active apty for IgE in a separate binding assay. tamer sequences. (J. Am. Chem. Soc. The isolated aptamers were specific for 2004, 126, 20–21) human IgE and did not interact with % Binding DNA
Systematic evolution of ligands by exponential enrichment (SELEX) is commonly used to identify DNA and RNA aptamers from combinatorial libraries; however, the technique suffers from selection biases, which can affect the quality of the aptamers. By adding a CE step to separate the binding sequences from the inactive ones, Michael Bowser and Shaun Mendonsa at the University of Minnesota have greatly improved the technique. CE-SELEX offers better enrichment and isolates higher affinity aptamers than conventional SELEX. Targets such as drugs, amino acids, or cells are bound to a column via a linker in conventional SELEX. Thus, aptamers are not exposed to the whole target surface and a DNA or RNA binding site may be obscured. Also, aptamers that bind targets with high affinity are difficult to isolate by SELEX because it is often not possible to release them from the column. Once aptamers are eluted from the column, the nucleic acid sequences are amplified by PCR and then added to the column
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Characterizing nanoparticle surfaces Two nanocrystals that appear to be the same
to particle aggregation, even small aggre-
two distributions differed in peak maximum,
in shape, size, size distribution, and crystal
gates in tiny amounts.
width, shape, and response to pH. The
structure may actually behave quite differ-
To determine whether AUC can be ap-
analysis revealed a different extent of pH-
ently because of differences in their surface
plied to the surface characterization of
dependent aggregation for the two sam-
structures. To better characterize nanoparti-
nanoparticles, Cölfen and colleagues per-
ples, presumably because of variations in
cle surfaces, Helmut Cölfen and colleagues
formed sedimentation velocity experiments
their surface properties.
at the Max Planck Institute of Colloids and In-
on two TiO2 nanocrystal suspensions. The
terfaces (Germany) and Bar-Ilan University
two sets of colloids were similar in size
can distinguish between particles with re-
(Israel) have turned to a technique called
(18-nm diam), size distribution, and crystal
spect to their surface structures as well as
analytical ultracentrifugation (AUC).
structure (anatase), but their surface struc-
their aggregation state. In the future, the re-
ture distributions differed.
searchers plan to complement AUC with a
AUC fractionates particles on the basis of their size, density, shape, and charge
Using AUC, the researchers determined
AUC appears to be a sensitive tool that
method such as flow field-flow fractiona-
and is used to determine sedimentation co-
the sedimentation coefficient distributions
tion, which determines size and charge dis-
efficients. The technique is highly sensitive
of the two samples as a function of pH. The
tributions. (Langmuir 2003, 19, 10,654–10,659)
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