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Biochemistry JoVE (Open Access) Citable · DOI

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

DOI: 10.3791/52545-v
What you'll learn
  • ✓ Select structure-switching aptamers using magnetic bead-based SELEX methodology
  • ✓ Monitor and amplify aptamer pools across iterative selection rounds
  • ✓ Implement colorimetric gold nanoparticle assay for target detection
  • ✓ Apply aptamer selection protocol to small molecule targets like cortisol
Protocol

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Difficulty
advanced
Total time
~5-7 days (multiple selection rounds with overnight incubations)
Biosafety
BSL-1

Steps

1
Prepare magnetic beads for selection

Prepare and condition magnetic beads that will be used to capture target-aptamer complexes during the selection process. This foundational step ensures consistent performance across selection rounds.

▶ 01:41
2
Perform first round of aptamer selection

Incubate aptamer library with target-coated magnetic beads under initial selection stringency conditions to enrich for binding aptamers. Isolate bound sequences from unbound pool.

▶ 04:03
3
Monitor and amplify between selection rounds

Quantify enriched aptamer pool and amplify via PCR to generate sufficient material for subsequent selection rounds. Assess progress toward structure-switching candidates.

▶ 05:36
4
Execute subsequent selection rounds with increased stringency

Repeat selection cycles with progressively higher stringency to isolate aptamers with improved target binding and structure-switching properties.

▶ 08:40
5
Validate aptamers with gold nanoparticle assay

Apply selected aptamers to a colorimetric gold nanoparticle-based assay to confirm structure-switching behavior and signal generation upon target binding.

▶ 09:51
6
Demonstrate aptamer selection using cortisol

Apply the complete protocol to cortisol as a model small molecule target, showing practical implementation of the structure-switching aptamer selection method.

▶ 10:33
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