In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target

Annamaria Ruscito1, Erin M. McConnell1, Anna Koudrina1, Ranganathan Velu1, Christopher Mattice1, Vernon Hunt1, Maureen McKeague2, Maria C. DeRosa1

1 Chemistry Department, Carleton University, Ottawa, Ontario, 2 Department of Health Sciences and Technology, ETH Zürich, Zurich
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.28
Online Posting Date:  December, 2017
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Aptamers, synthetic oligonucleotide‐based molecular recognition probes, have found use in a wide array of biosensing technologies based on their tight and highly selective binding to a variety of molecular targets. However, the inherent challenges associated with the selection and characterization of aptamers for small molecule targets have resulted in their underrepresentation, despite the need for small molecule detection in fields such as medicine, the environment, and agriculture. This protocol describes the steps in the selection, sequencing, affinity characterization, and truncation of DNA aptamers that are specific for small molecule targets. © 2017 by John Wiley & Sons, Inc.

Keywords: aptamer; DNA; high throughput sequencing; in vitro selection; SELEX; small molecule

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Isolating a Functionally Enriched Aptamer Pool by Column Selection
  • Alternate Protocol 1: Capture‐Selex
  • Support Protocol 1: DNA Amplification, Separation, and Isolation
  • Support Protocol 2: DNA Aptamer Sequencing by MiSeq
  • Basic Protocol 2: Assessing Aptamer Affinity
  • Support Protocol 3: Aptamer Truncation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Isolating a Functionally Enriched Aptamer Pool by Column Selection

  • Household bleach diluted 1:10 (to 0.5% hypochlorite) and/or 70% ethanol
  • Bead slurry (e.g., Pierce TM NHS‐Activated Agarose Slurry; Thermo Fisher, cat. no. 26200; also see Critical Parameters)
  • Bead slurry with target covalently bound (see Critical Parameters for conjugation of molecules to beads)
  • SELEX buffer 1 (see recipe)
  • Library sequence: 1 nmol of 5′‐labeled (6‐FAM; 6‐fluorescein amidite) DNA library (for round 1, 500 pmol for rounds 2 and 3, 200 pmol onwards), see example of library design in protocol 3: part of the original DNA library is used to start the first round of SELEX (in this protocol or or protocol 2) and the other part is set aside to be used as a positive control for PCR in protocol 3
  • 7 M urea (store up to several months at room temperature), preheated to 90°C
  • PCR Workstation (e.g., AirClean® Systems AC600 Series PCR Workstations combining ISO 5 HEPA‐filtered air with UV‐light irradiation)
  • Corning™ Costar™ Spin‐X™ Centrifuge Tube Filters (Fisher Scientific, cat. no. 07‐200‐385) or equivalent sample prep unit with a 0.22‐µm cellulose acetate filter
  • 1.5‐ml polypropylene microcentrifuge tubes
  • Analytical balance
  • Sorvall Legend microcentrifuge (Thermo Fisher)
  • 90°C heat block (Sigma Aldrich, cat. no. SKU Z381519)
  • Vortex‐Genie® 2 (Scientific Industries, Inc., cat. no. 0K‐0500‐902)
  • Fluorometer (e.g., HORIBA Jobin Yvon)
  • 45‐µl Hellma® fluorescence cuvette, ultramicro (Sigma‐Aldrich, cat. no. SKU Z802433)
NOTE: DNA library and primers can be synthesized by standard phosphoramidite chemistry or purchased from a commercial oligonucleotide supplier, such as Integrated DNA Technologies (IDT). The library and primer sequences can be found in protocol 3.

Alternate Protocol 1: Capture‐Selex

  Additional Materials (also see protocol 1)
  • 1 nmol of 5′‐labeled (6‐FAM) DNA library containing capture probe binding region (for round 1,500 pmol for rounds 2 and 3, 200 pmol onwards)
  • 5 nmol of 5′‐biotin‐labeled capture probe [7 to 15 bases long with either a poly (A), poly (T) or hexaethylene glycol (HEGL) spacer] for immobilization
  • SELEX buffer 2 (see recipe)
  • Avidin‐agarose beads (Sigma‐Aldrich)
  • 95°C heat block (Sigma Aldrich, cat. no. SKU Z381519)
  • Shaker
  • Amicon Ultra‐0.5 ml centrifugal filters (NMWL, 3 kDa; EMD Millipore, cat. no. UFC5003BK)
  • Savant DNA SpeedVac Concentrator (Thermo Fisher)
  • Fluorometer (e.g., HORIBA Jobin Yvon)

Support Protocol 1: DNA Amplification, Separation, and Isolation

  • PCR buffer (see recipe)
  • 25 mM MgCl 2
  • 0.2 mM dNTP mix (0.2 mM dATP, dCTP, dGTP, and dTTP)
  • Forward primer: 5′‐FAM‐ATCCAGAGTGACGCAGCA
  • Reverse primer: 5′ ‐poly‐dA20‐HEG‐ACTAAGCCACCGTGTCCA
  • 6‐FAM is 6‐fluorescein phosphoramidite and HEG is hexaethyleneglycol spacer phosphoramidite 18. Both 6‐FAM and HEG can be purchased from Glen Research (cat. nos. 10‐1963 and 10‐1918, respectively).
  • Taq polymerase (Bioshop Canada, cat. no. TAQ001.500)
  • Template DNA (DNA pool that resulted from the latest SELEX round using protocol 1 or protocol 2) in deionized water
  • Original DNA library (DNA that has never gone through a SELEX round) as positive control: e.g., 5′‐TGGTGGCTGTAGGTCA‐N40‐GAGCATCGGACAACG (see primer sequences above)
  • Urea
  • 40% acrylamide stock (1:19 bisacrylamide:acrylamide)
  • 5× TBE buffer (see recipe)
  • Ammonium persulfate (APS)
  • N,N,N',N'‐tetramethylethylenediamine (TEMED)
  • Formamide
  • 1.5‐ml and 2‐ml microcentrifuge tubes
  • 200 µl sterile, nuclease‐free PCR tubes
  • Thermocycler (Eppendorf)
  • Savant SpeedVac AES2010 concentrator
  • 25‐ml beaker
  • Whatman No. 1 filter paper
  • Gel electrophoresis equipment: e.g., Hoefer SE 600 Chroma Standard dual‐gel electrophoresis unit
  • Plastic wrap
  • 20 × 20 thin‐layer chromatography (TLC) plate with fluorescent indicator
  • Multiimage light cabinet with camera (Alpha Innotech)
  • 50‐ml tubes
  • Amicon Ultra‐0.5 ml centrifugal filters (NMWL, 3 kDa; EMD Millipore, cat. no. UFC5003BK)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (Ellington & Pollard, )

Support Protocol 2: DNA Aptamer Sequencing by MiSeq

  • Forward primer(s) with appropriate adapter sequence (see Table 28.0.1)
  • Reverse primer(s) with appropriate adapter sequence (see Table 28.0.1)
  • PhiX Control V3 (Illumina, cat. no. FC‐110‐3001)
  • MiSeq Reagent Kits v2 (300 or 500 cycles depending on the template + elongated primer length; use 500 if the PCR amplicon is greater than 150 base pairs; Illumina, cat. no. MS‐102‐2002 or MS‐102‐2003)
Table h.8.1   MaterialsExamples of Primers Compatible with MiSeq Illumina Sequencing Technology a

Primer name 5′→3′

 aFixed primer regions are shown in normal text. The forward and reverse primer sequences that are specific to the template used should be inserted at the 3′‐end of the fixed regions where appropriate. Random nucleotide spacers are indicated by a bold N, and the barcodes that are complementary to the index adapters are bold and underlined.
  • Nanodrop 2000 (Thermo Fisher) or other method for DNA quantification
  • Illumina MiSeq System (Illumina)
  • Aptamer sequencing analysis software package (see Critical Parameters)
  • Additional reagents and equipment for DNA amplification, separation, and isolation ( protocol 3)

Basic Protocol 2: Assessing Aptamer Affinity

  • 10 µM aptamer candidate DNA
  • 10.5 to 11 nM AuNP solution (red, 10‐ or 15‐nm diameter) from NanoComposix or fabricated using the Turkevich method (Turkevich, Stevenson, & Hillier, )
  • Target
  • 0.25 mM NaCl
  • Bead slurry (e.g., NHS‐Activated Magnetic Beads; Pierce™, cat. no. 88826, see Critical Parameters for more information)
  • Bead slurry with target covalently attached
  • SELEX buffer 1 (see recipe)
  • 7 M urea, heated to 90°C
  • 90°C heat block (Sigma‐Aldrich, cat. no. SKU Z381519)
  • DynaMag™‐2 Magnet (Thermo Fisher, cat. no. 12321D)
  • Vortex‐Genie® 2 (Scientific Industries, Inc. Cat. No. 0K‐0500‐902)
  • Additional reagents and equipment for quantifying fluorescence signal ( protocol 1)

Support Protocol 3: Aptamer Truncation

  • 50 μM 5′‐fluorescein‐labeled DNA (truncated aptamer designs) dissolved in deionized water
  • Aptamer target
  • DNase I reaction buffer (see recipe)
  • DNase I (New England Biolabs)
  • 0.5 M EDTA, pH 8
  • Formamide
  • Computer with RNAstructure and GraphPad Prism installed
  • Vortex‐Genie® 2 (Scientific Industries, Inc. Cat. No. 0K‐0500‐902)
  • Heat block (Sigma Aldrich, cat. no. SKU Z381519)
  • Gel electrophoresis unit (e.g., a Hoefer SE 600 Chroma Standard dual gel electrophoresis unit)
  • Multiimage light cabinet with camera (Alpha Innotech) with SpotDenso software
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (PAGE; Ellington & Pollard, )
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