NGS for the identification of DNA and RNA aptamers
A DNA or RNA aptamer is a nucleic acid ligand that binds to proteins, peptides or small molecule targets with high affinity and specificity. Aptamers are routinely isolated from combinatorial single stranded libraries by applying the SELEX method (Systematic Evolution of Ligands by EXponential enrichment). Aptamer RNA or DNA sequences fold into rigid structures that bind their target by complementary shape recognition.
Next-generation sequencing (NGS) promises to transform the aptamer’s identification process significantly. SELEX is an in vitro selection procedure that uses iterative cycles of selection and amplification (typically 6-15) for identifying aptamers addressing the target of interest.
The SELEX process for the identification of DNA/RNA aptamers:
It is frequently necessary to combine SELEX against the actual target with one or more additional methods of selection such as counter selection, competitive elution, etc. in order to shift the population of selected DNA or RNA aptamers to those with the desired binding characteristics. This makes SELEX a quite laborious method. Aptamer RNA ligands that lack the desired binding characteristics are a frequent result. AptaIT’s next-generation sequencing bioinformatics platform subsumes more than 20 years of SELEX expertise and addresses such relevant issues.
DNA/RNA aptamer identification by NGS data analysis:
Next-generation sequencing improves innovative SELEX methods by:
- Quality control of combinatorial SELEX libraries by NGS data analysis
- More effective identification of DNA and RNA aptamers in early selection cycles
- Optimization of DNA and RNA aptamer lead candidates
Quality control of aptamer libraries
The outcome of biopanning experiments is strongly dependent the quality of the initial phage display library , which is used as the source to extract the best binders. The more the distribution of amino acids is in accordance with the specifications, the better is the library’s diversity and the chance to select better binders. Next-generation sequencing of the initial phage display libraries is a very helpful method to receive solid statistical data on the distribution of amino acids in defined positions or frequencies of motives in a starting library. The sequencing data can be examined on the level of nucleotides as well as amino acids.
Identification of DNA and RNA aptamers
AptaIT’s bioinformatics approach is able to identify hundreds to thousands of different aptamers by analyzing NGS data from different SELEX cycles. Additionally, related sequences are sorted into a reasonable number of families to allow an effective analysis of representatives in an affinity-based or functional assay. Testing just the most frequent candidates like in conventional SELEX may not be the right idea as the most dominant aptamer sequences will not be the ones with the optimal specificity, affinity and functionality.
NGS data analysis improves SELEX by:
- Reducing the number of selection cycles
- Identification of rare but valuable DNA or RNA aptamers
- Subtracting false positives and contaminations
- Successfully employing difficult selections, e.g. negative selections or cells as targets
- Tracking ligands over comparative selection experiments
Aptamer lead optimization
Next-generation sequencing data of DNA and RNA aptamer sequences can also be used for a deep similarity analysis once sequence families of aptamers with the expected properties have been identified. We use the already available sequence information contained in the entire family to identify conserved sequence motives, minimal binding motives, and variable positions. The unprecedented amount of data provides the best information available with respect to lead optimization and advanced patenting strategies.
Download the DNA and RNA aptamer application note for a more detailed overview. AptaIT is providing innovative solutions to exploit the full potential of next-generation sequencing data analysis and SELEX: Custom NGS data analysis services and our software platform COMPAS.