Cell-SELEX

Cell-based SELEX is a powerful approach for identifying aptamers that can be selectively bind to specific cell-surface proteins, transmembrane receptors or a particular target cell population. Those cells could be bacterial strains or mammalian cells.

Cell-SELEX methodology relies on the differences between the target cells population (positive cells that express the target of interest) and the control cell population (negative cells or counter-selection cells that do not express the target protein). Therefore, the selection procedure generally includes positive selection against the target cells and counter selection against non-target cells to remove nonspecific binding.

Cell-SELEX Advantages

• • High Specificity and Affinity: Aptamers selected through Cell-SELEX can bind to their targets with high specificity and affinity.
  • Versatility: This method can be used to target a wide range of cells, including cancer cells, bacteria, and viruses.
  • No Need for Prior Knowledge: Unlike traditional methods, Cell-SELEX does not require prior knowledge of the target’s molecular structure.

Cell-SELEX Applications

• • Diagnostics: Aptamers can be used as molecular probes to detect disease biomarkers.
  • Therapeutics: Aptamers can be developed into therapeutic agents that specifically target diseased cells.

Cell-SELEX Main Steps

1. 1. Counter selection by incubating library with negative cells that do not express the target proteins;
   2. Positive selection by incubating recovered unbound sequences with positive cells expressing the target protein.
   3. Recovery of target-bound sequences
   4. Re-amplification of recovered sequences and make new pool for the next selection round.
   5. After 5-12 selection rounds, individual aptamer candidates are identified by Sanger Sequencing or Deep Sequencing and bioinformatics analysis. 

Cell-Internalization SELEX

For delivering secondary reagents, such as aptamer-drug, aptamer-siRNAs and aptamer-nanoparticle conjugates, into the target cells, cell internalized aptamers is best choice through cell-internalization SLEX technologies.

Many ligands of cell surface proteins are efficiently internalized after binding their protein targets on the cell surface. Thus, we could identify aptamers not only bind a particular target protein on the surface of cells, but also subsequently internalized into the cell.

Cell-Internalization SELEX Main Steps

1. 1. The starting DNA or RNA aptamer library is pre-cleared by incubating the library with non-target cells (The non-target cells are identical to the target receptor-expressing cells, except that they lack the target receptor).
   2. Those DNAs or RNAs that do not bind or internalize into the non-target cells are then transferred to the target cells. Unbound and surface-bound DNAs or RNAs are discarded and internalized DNAs or RNAs recovered.
   3. The recovered DNAs or RNAs are amplified using PCR or RT-PCR and the resulting amplified dsDNA digested to ssDNA or in vitro transcribed to RNAs for the subsequent round of selection.
   4. After 5-12 selection rounds (the selection is terminated when the library reached 90% sequence convergence), individual aptamer candidates are identified by Sanger Sequencing or Deep Sequencing and bioinformatics analysis. 

Aptamer Development through Cell-SELEX

Phase I: Library Enrichment via Cell-SELEX

Iterative rounds (5-12 rounds) of screening an initial random library against target cells for positive selection, and negative and/or counter-selection screening against non-target cells.

Criteria: Enrichment 8-30%.

Deliverables: full enrichment progress report

Timeline: 2-3 weeks

Phase II: Sanger Sequencing & Bioinformatic Analysis

Last round of positive binding candidates cloning into sequencing vector for polyclonal preparation, and picking up to 48 clones for Sanger sequencing.

Deliverables: Aptamer candidate sequence information, sequence alignment, family cohort, secondary structure analysis, docking simulation et al..

Timeline: 1-2 weeks

Phase III: NGS Sequencing & Bioinformatic Analysis (optional)

High throughput sequencing (NGS) of last round and representative rounds of positive binding and negative binding, bioimformatic analysis to identify the potential top 5-10 aptamer candidates. Ranked according to the ratio of their representation in the positive round as compared to control population(s).   

Deliverables: NGS analysis full report

Timeline: 2-3 weeks

Phase IV: Aptamer Candidate Synthesis, Affinity Determination and Specificity Assessment

Each aptamer candidate (up to 8) is synthesized and quantitatively tested for Kd affinities.

Deliverables: 10 nmol each aptamer candidates, and full assay report showing K_D value and specificity for each aptamer candidates.

Timeline: 2-3 weeks

Phase V: Aptamer Optimization & Assay Development

Depends on the criteria set by clients, the cost and timeline will be different.

Deliverables: Full assay development report and 10m nmol optimized aptamers.

Timeline: 2-4 weeks

Packages for your Cell-SELEX project

	Basic Package	Standard Package	Premium Package	Premium plus Package
Phase I: Library Enrichment via Cell-SELEX
Phase II: Sanger Sequencing & Bioinformatic Analysis
Phase III: NGS Sequencing & Bioinformatic Analysis
Phase IV: Aptamer Candidate Synthesis, Affinity Determination and Specificity Assessment
Phase V: Aptamer Optimization & Assay Development
Package Cost*	inquiry	inquiry	inquiry	inquiry

1) Fusion BioLabs guarantees the services;

2) Fusion BioLabs owns its proprietary processes of aptamer development. However, the client owns full rights to the aptamers Fusion BioLabs developed for the client;

3) For multiple targets aptamer development, please contact us for special pricing.   

References

1. Thiel WH, et al. (2015) Cell-internalization SELEX: method for identifying cell-internalizing RNA aptamers for delivery siRNAs to target cells. Methods in Molecular Biology, 1218: 187-199.

2. Zhou J and Rossi J (2016) Evolution of cell-type-specific RNA aptamers via live fell-based SELEX.  Methods in Molecular Biology, 1421: 191-214.