Multiplexed microcolumn-based process for efficient selection of RNA aptamers.

Area of Science:

• Biotechnology
• Molecular Biology
• Biochemistry

Background:

• Aptamer discovery is crucial for molecular diagnostics and therapeutics.
• Current methods can be time-consuming and require large sample volumes.
• Efficient selection of aptamers against multiple targets remains a challenge.

Purpose of the Study:

• To develop a reusable microcolumn and process for efficient, multiplexed nucleic acid aptamer discovery.
• To optimize aptamer selection using microcolumns with limited resin volumes.
• To validate the multiplex approach for identifying high-affinity aptamers against specific protein targets.

Main Methods:

• Utilized a reusable microcolumn with affinity chromatography resin for aptamer selection.
• Optimized selection by monitoring enrichment of a known aptamer (GFPapt) against green fluorescent protein (GFP).
• Employed a multiplex selection strategy for two human heat shock factor (hHSF) proteins (hHSF1 and hHSF2) combined with high-throughput sequencing.

Main Results:

• Achieved >10^8-fold enrichment of GFPapt in three selection rounds with minimal non-specific binding.
• Identified and isolated specific aptamers for hHSF1 and hHSF2 after five rounds of multiplex selection.
• Confirmed high-affinity binding (KD < 20 nM) of selected aptamers to their respective targets using biophysical assays.

Conclusions:

• The microcolumn and multiplex approach enable high-throughput and efficient aptamer selection for multiple targets.
• This method significantly reduces the volume of affinity resin required, maximizing target-binding sequence enrichment.
• The developed protocol is effective for discovering specific, high-affinity aptamers against complex protein targets.