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Systematic Evolution of Ligands by Exponential Enrichment


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Systematic evolution of ligands by exponential enrichment (SELEX) is a very powerful tool for identifying the binding sites of a protein on RNA. Lifeasible is at the forefront of the biotechnology revolution using SELEX technologies. We provide high-quality SELEX analytical service for customers from both academia and industry. We strive to give our customers the newest protocol and the most reliable SELEX analytical data.

SELEX depends on the fact that RNA binding proteins are capable of selecting high-affinity RNA ligands from large randomized pools of different RNAs. SELEX assay is performed several rounds and each round can increase the enrichment of target RNAs binding with a specific protein. Therefore, SELEX is extremely useful for determining preferred RNA binding sites for the protein of interest. SELEX generally includes the following steps (Figure 1):

Systematic-evolution-of-ligands-by-exponential-enrichment
Figure 1. The workflow of the SELEX assay (Popova et al., 2015)

1. Generation of double-stranded DNA templates from an oligonucleotide library for transcription

The resulting double-stranded templates contain the restriction sites for subsequent cloning, the T7 promoter, and a randomized sequence.

2. Transcription of the double-stranded DNA templates to obtain RNA

The RNA library is obtained by transcription which is carried out with T7 polymerase.

3. Protein–RNA binding and selection

Following with transcription, the RNA library is allowed to bind with a protein of interest.

4. cDNA synthesis by reverse transcription

Bound RNAs are purified and converted to a cDNA library with the help of a 3′ primer and reverse transcriptase.

5. Generation of double-stranded DNA template for subsequent rounds

Double-stranded DNA templates are produced by PCR with the 5′ and 3′ primers using the cDNA pool. And the rounds are repeated until the enrichment in RNAs that bind with the protein reaches 90%.

6. Determine the sequence recognized by the protein

The resulting nucleotide sequences are cloned in a vector and sequenced.

Thanks to our excellent group of professionals and Hi-Tech facilities, we now have serviced customers from many countries. Lifeasible provides one-stop SELEX analytical service from RNA library preparation, RNA binding, sequencing to data analysis. Lifeasible has built-up leading scientific track records in RNA-protein interaction study. If you have any question, please contact us anytime for assistance in business days. Our experienced project managers will provide you professional support to ensure the success of your project.

Reference

  1. Popova V. V.; et al. Methods to study the RNA–protein interactions. Molecular Biology, 2015, 49 (3): 418–426.
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