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Random Nucleic Acid Libraries for SELEX (in vitro Selection) in Aptamer Development

in vitro Selection (SELEX)

The word aptamer comes from the Greek word "aptus", meaning "to fit". Aptamers are obtained through a process called in vitro selection or SELEX (Systematic Evolution of Ligands by Exponential Enrichment). The objective of an in vitro selection is to screen a large library of sequences (e.g. 1015 molecules) to identify an aptamer sequence that has a desired property. This is often binding to a target protein or small molecule, although in other applications, researchers use SELEX to evolve aptamers with a desired catalytic activity. Three separate laboratories were simultaneously working on the development of this technology: Larry Gold and Craig Tuerk at the University of Colorado Boulder, Jack Szostak and Andy Ellington at Massachusetts General Hospital and Gerald Joyce at the Scripps Institute in La Jolla, CA. The Colorado group was granted a patent in 1993.

During selections for aptamers that bind a target molecule, the work flow is as follows. A large library of sequences is combined with a target molecule that is often immobilized. A small subset of the library adopts conformations that mediate tight binding to the target. The majority of sequences do not bind and are subsequently washed away. The binding aptamers are released and amplified by PCR. These three steps of selection, partitioning and amplification are repeated numerous times until aptamers with the desired binding affinities are identified. RNA or DNA libraries are used for evolution of RNA or DNA aptamers, respectively.

Random Mutagenesis

Often, a random mutagenesis step is added to the amplification step of the SELEX cycle. This mutagenesis step diversifies the selected population of sequences during the workflow, allowing for access to sequences with stronger binding affinities. Random mutagenesis is performed by modification to the PCR conditions. Approaches to random mutagenesis have included a combination of unbalanced dNTP ratios and manganese chloride as well as mutagenic dNTP analogs, such as 8-oxo-dGTP and dPTP. To complement our stocked random libraries for use in SELEX, we also offer a Mutagenesis dNTP Mix, which contains the mutagenic dNTP analogs described and characterized by Zaccolo et. al.

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Custom Libraries

TriLink is capable of producing almost any library, including different lengths of random sequence, other chemical modifications, different primer binding sites and minimal primer or primer-free libraries. These libraries can be made with varying base composition ratios. Our basic service entails synthesis with mixtures of bases that are predicted to yield the desired ratios. The actual ratio is determined by base composition analysis. If a precise final mixture is required, the experimentation necessary to produce the custom mix desired can be conducted.

Doped libraries are available as well. Doped libraries are used when starting from a known aptamer sequence and selecting for motifs with improved or altered properties. For example, a given position in a doped library could contain the starting sequence at 70% frequency, while introducing each of the other three nucleotides at 10% frequency each (30% doping). For a 30 nt starting aptamer doped at a frequency of 30%, each member of the resultant library will contain an average of 9 mutations relative to the parent sequence (30 nt * 0.30 mutations/nt). Key parameters to consider in the preparation of a doped pool include effectively randomizing the starting motif without deviating too far from the starting aptamer sequence. In addition to more traditional library constructs, TriLink can also prepare your doped library of choice.

The entire listing of modified bases available from TriLink can be used to prepare libraries, which opens up the potential structure space for aptamer development tremendously. Note that if multiple rounds of selection are to be performed, the modifications chosen need to be incorporated by the DNA polymerase used in PCR or the RNA polymerase used for transcription.

The following modifications have been shown to be transcribed (with varying efficiencies) by wild type T7 RNA polymerase:

ATP analogs CTP analogs GTP analogs UTP analogs
N6-Methyl-ATP 5-Methyl-CTP 7-Deaza-GTP 5-Bromo-UTP
8-Azido-ATP 5-Bromo-CTP Alpha-Thiol GTP 5-Iodo-UTP
7-Deaza-ATP 5-Aminoallyl-CTP   5-Methyl-UTP
2-Aminopurine rTP Alpha-Thiol CTP   Biotin-16-UTP
Alpha-Thiol ATP Biotin-16-CTP   Pseudo-UTP
      Alpha-Thiol UTP

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