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Patrick C.H. Lo, Ph.D. and Kristie Nybo, Ph.D.
BioTechniques, Vol. 51, No. 6, December 2011, p. 365
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Aptamers are nucleic acid oligomers that bind target molecules with high affinity and selectivity. They are usually identified through a technique called systemic evolution of ligands by exponential enrichment (SELEX), wherein a library of random oligonucleotide sequences is initially exposed to the target molecule, after which binding oligonucleotides are recovered, PCR amplified, and then subjected to further rounds of selection. Following this selection process, the isolated oligonucleotides are cloned and sequenced, identifying the high-affinity aptamers against the target molecule. While this well-established method has proven to be effective in aptamer identification, it can also be time consuming and laborious owing to those numerous rounds of oligonucleotide selection. In this month's issue, J. Scolnick and colleagues at The Scripps Research Institute (La Jolla, CA) describe their novel approach to identifying high-affinity DNA aptamers after only a single round of selection. The authors make use of high-throughput sequencing technology and bioinformatics analysis to identify variable k-mer length subsequences that are enriched in a sequenced library of the selected oligonucleotides. The technique, named aptamer selection by k-mer analysis of sequences (ASKAS), was validated by identifying aptamers that bind thrombin. A library of random 33-mer oligonucleotides was initially subjected to negative selection by pre-binding to blank magnetic beads. Unbound oligonucleotides were then incubated with thrombin-coated magnetic beads. Any oligonucleotides that did not bind the thrombincoated beads were removed with multiple washes while bound oligonucleotides were directly PCR amplified and sequenced using an Illumina GA2x alongside a non-thrombin-selected (ie. naïve) library. For both libraries, an aptamer known to bind thrombin was added as a positive control. Initial sequence analysis indicated that the positive control aptamer as well as other 33-mer oligonucleotide sequences were enriched in the thrombin-selected library compared to the naïve library. K-mer analysis was carried out to identify subsequences within the thrombin-selected 33-mer oligonucleotides responsible for thrombin binding. Of the top five 15-mer and 16-mer k-mers the authors focused on, nine had dissociation constants in the nanomolar or micromolar range, with two in the tens of nanomolar range; one of these was previously identified, whereas the other was novel. Taken together, the authors clearly demonstrate that ASKAS is a useful approach for aptamer identification.

See “Aptamer selection by high-throughput sequencing and informatic analysis”.

Plugging the Leak

Since their development, reporter assays have proven to be critical tools in many molecular biology studies. For example, reporter assays have been used as indicators of promoter activity in a variety of backgrounds, from cells in culture to plants, and even in vivo using transgenic animals. In such cases, the expression of a fluorescent protein (FP) reporter can be used to precisely target specific cell types or time periods, or elucidate cell lineages. Validating that FP expression reflects the specificity of endogenous gene activity in vivo can be accomplished using flow cytometry, which is also valuable for querying other molecular properties of FP-labeled cells under various experimental conditions. However, this approach requires the labeling of FP expressing cells with antibodies against proteins of interest, a challenge as the staining procedure required to prepare the cells for flow cytometry often causes a loss of cytosolic FP signal. In the course of their research, Grupillo et al. encountered this problem and hypothesized that the permeabilization step might allow leakage of cytosolic FPs from the cells. Adding an additional fixing step prior to permeabilization prevented leakage of the FP from the cells, allowing Grupillo and his colleagues to detect its signal after completion of the staining procedure. However, this change prevented detection of subsequent antibody signals. Other researchers have avoided this issue by isolating FP-expressing cells using fluorescence activated cell sorting prior to antibody staining. But this alternative is time consuming, impractical when working with rare cells, and does not allow simultaneous detection of both signals. Writing in the current issue of BioTechniques, Grupillo et al. now describe a modified staining method that enables simultaneous detection of both nuclear proteins and cytosolic FP molecules. By employing an optimized additional fixing step prior to permeabilization and adding saponin to the permeabilization buffer, the authors were able to prevent FP leakage from cells during staining, as well as successfully stain for various cell surface markers. This modified procedure should better facilitate the validation of reporter assays in FP-expressing transgenic animals and enable co-localization studies involving cytosolic FP and flow cytometry.

See “An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells”.