In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

/pdf/in-vitro-selection-of-functional-nucleic-acids-2gyp6kaqym.pdf

In vitro selection of functional nucleic acids.

Aptamers are single-stranded DNA or RNA molecules that can bind target molecules with high affinity and specificity. The conformation of an aptamer usually changes upon binding to its target analyte, and this property has been used in a wide variety of sensing applications, including detection based on fluorescence intensity, polarization, energy transfer, electrochemistry or color change. Colorimetric sensors are particularly important because they minimize or eliminate the necessity of using expensive and complicated instruments. Among the many colorimetric sensing strategies, metallic nanoparticle-based detection is desirable because of the high extinction coefficients and strong distance-dependent optical properties of the nanoparticles. Here, we describe a protocol for the preparation of aptamer-linked gold nanoparticle purple aggregates that undergo fast disassembly into red dispersed nanoparticles upon binding of target analytes. This method has proved to be generally applicable for colorimetric sensing of a broad range of analytes. The time range for the entire protocol is ∼5 d, including synthesis and functionalization of nanoparticles, preparation of nanoparticle aggregates and sensing.

Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes

http://dbkgroup.org/Papers/davey_kell_micrev96.pdf

Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analyses.

Aptamers are short single-stranded oligonucleotides that are capable of binding various molecules with high affinity and specificity. When the technology of aptamer selection was developed almost a quarter of a century ago, a suggestion was immediately put forward that it might be a revolutionary start into solving many problems associated with diagnostics and the therapy of diseases. However, multiple attempts to use aptamers in practice, although sometimes successful, have been generally much less efficient than had been expected initially. This review is mostly devoted not to the successful use of aptamers but to the problems impeding the widespread application of aptamers in diagnostics and therapy, as well as to approaches that could considerably expand the range of aptamer application.

/pdf/aptamers-problems-solutions-and-prospects-1n6ccfvlsm.pdf

Aptamers: Problems, Solutions and Prospects

SELEX (systematic evolution of ligands by exponential enrichment) is a process that involves the progressive purification from a combinatorial library of nucleic acid ligands with a high affinity for a particular target by repeated rounds of partitioning and amplification. With the development of aptamer technology over the last decade, various modified SELEX processes have arisen that allow various aptamers to be developed against a wide variety of molecules, irrespective of the target size. In the present review, the separation methods used in such SELEX processes are reviewed.

Methods developed for SELEX.

Publisher Summary Nucleic acid sequences can fold into a huge variety of shapes. Some of these shapes can form complexes, with chemically complementary compounds. The development of more sophisticated in vitro selection methods has made possible the isolation of nucleic acid-binding species (aptamers) that can tightly and specifically interact with a variety of proteins and small molecules. Specifically, in vitro selection has been used to probe interactions between nucleic acids and proteins that normally bind nucleic acids. Subsequently, in vitro selection has been used both to define natural nucleic acid-binding sites for proteins and to identify novel high-affinity sites for these proteins. In vitro selection experiments have not only increased the understanding of the biology of nucleic acid-binding proteins, but have also identified aptamers that can potentially act as therapeutically useful “decoys.” Surprisingly, in vitro selection can also be used to isolate aptamers that can specifically interact, with proteins that are not normally thought to bind nucleic acids.

In vitro selection of nucleic acid aptamers that bind proteins.

Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies.

Dissecting protein:protein interactions between transcription factors with an RNA aptamer.

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In vitro selection of nucleic acid aptamers that bind proteins.

Dissecting protein:protein interactions between transcription factors with an RNA aptamer.