Rolling Circle Amplification with Chemically Modified Nucleoside Triphosphates

Marcel Hollenstein1, Masad J. Damha2

1 Department of Structural Biology and Chemistry, Pasteur Institute, Paris, 2 Department of Chemistry, McGill University, Montreal, Quebec
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.26
DOI:  10.1002/cpnc.17
Online Posting Date:  December, 2016
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Modified nucleoside triphosphates (dN*TPs) represent facile and versatile precursors for the introduction of chemical diversity into nucleic acids. While dN*TPs have been utilized in a plethora of practical applications, very little attention has been devoted to the assessment of their compatibility with isothermal amplification strategies. In this context, rolling circle amplification (RCA) is a wide‐spread enzymatic replication method in which small single‐stranded DNA (ssDNA) circles serve as templates in primer extension reactions yielding very long, ssDNA products. RCA is a pivotal tool for the generation of biosensor and diagnostic devices and is currently evaluated for its usefulness to create novel drug delivery systems. This unit describes the experimental procedures for the synthesis of modified RCA products using dN*TPs bearing chemical alterations at any possible location of the nucleosidic scaffold. Two ligation methods are presented for the generation of the DNA nanocircles that serve as templates for RCA, followed by a description of the RCA method itself and an assessment of the nuclease resistance of the ensuing products. © 2016 by John Wiley & Sons, Inc.

Keywords: rolling circle amplification; nucleoside triphosphates; polymerases; modified DNA; drug delivery systems

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of DNA Nanocircles via Standard Ligation
  • Alternate Protocol 1: Preparation of DNA Nanocircles via CircLigase Ligation
  • Support Protocol 1: Exonuclease Treatment of the Circular Templates
  • Basic Protocol 2: Rolling Circle Amplification with Modified Nucleoside Triphosphates
  • Support Protocol 2: Nuclease Resistance
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of DNA Nanocircles via Standard Ligation

  • 40% acrylamide/bis‐acrylamide solution (19:1, w/w, electrophoresis purity reagent; Serva Electrophoresis)
  • 10% (w/v) ammonium persulfate (APS) solution
  • BSA (Promega)
  • Bromophenol blue
  • EDTA
  • Ethanol (EtOH)
  • Formamide
  • Elution solution (1 mM in triethylamine [NEt 3], 1 wt. % LiClO 4, pH 8: Dissolve 1 g LiClO 4 [Sigma‐Aldrich] in 50 mL autoclaved Milli‐Q water; add 13.9 µL NEt 3 and adjust pH to 8 with HCl or NaOH; make up solution to 100 mL final volume with autoclaved Milli‐Q water.)
  • 10× TBE buffer (Sigma‐Aldrich or preparation by standard recipe; see appendix 2A)
  • T4 DNA ligase (Promega)
  • 10× T4 DNA ligase buffer (300 mM Tris·Cl, pH 7.8; 100 mM MgCl 2; 100 mM DTT; and 10 mM ATP)
  • Sephadex G10 (Sigma‐Aldrich)
  • Xylene cyanol FF
  • 5′‐Phosphate‐CTTGGTCTACTGGAG‐N 20‐CTACGGATTGC 1 (DNA template, see Strategic Planning; Microsynth)
  • 5′‐TAGACCAAGGCAATCCGTA (splint oligonucleotide, see Strategic Planning; Microsynth)
  • Urea (Apollo Scientific)
  • Centrifuge
  • Comb (8 wells)
  • Gel plates (20 × 20 cm)
  • Handheld UV lamp (254 nm)
  • Heating block
  • PAGE electrophoresis apparatus
  • 50‐mL plastic syringe
  • Power supply
  • 1.5‐mL screw‐top vials
  • Spacers (1 mm thick)
  • ThermoMixer
  • UV‐active thin‐layer chromatography plate (0.25 mm, UV 254; Macherey‐Nagel)
  • UV/vis spectrophotometer
  • Vacuum concentrator
  • Vortex mixing device

Alternate Protocol 1: Preparation of DNA Nanocircles via CircLigase Ligation

  • γ‐32P‐ATP (Hartmann Analytic)
  • Circular ssDNA templates (see Basic Protocols 1 and protocol 2Alternate Protocol)
  • DNA primers:
    • 5′‐TAGACCAAGGCAATCCGTA 3 (for circular template 1)
    • 5′‐CTAACCCTAACCCTAACC 4 (for circular templates 2 to 4)
  • Modified dNTPs:
    • 1 to 3 (base‐modified triphosphates; synthesized according to Hollenstein, )
    • 2′‐Deoxy‐2′‐fluoro‐β‐D‐arabinonucleosides (FANA) 5 (sugar‐modified triphosphates; prepared according to Peng and Damha, )
    • 4 (base‐modified triphosphate; TriLink BioTechnologies, cat. no. N‐2011)
    • 6 (sugar‐modified triphosphates; TriLink BioTechnologies, cat. nos. N‐1007 [for 2′‐F‐dATP]; N‐1008 [for 2′‐F‐dCTP]; N‐1055 [for 2′‐F‐dTTP])
    • 7 (phosphate modified; TriLink BioTechnologies, cat. nos. N‐8001 [for 1‐thio‐dATP]; N‐8002 [for 1‐thio‐dCTP]; N‐8004 [for 1‐thio‐dTTP])
  • N m DNA polymerase (New England Biolabs)
  • 10× Thermopol buffer
  • T4 PNK (Thermo Fisher Scientific)
  • 10× T4 PNK buffer (forward reactions; 500 mM Tris·Cl, pH 7.6; 100 mM MgCl 2; 50 mM DTT; 1  mM spermidine)
  • Stop solution (see protocol 1)
  • Urea
  • 10× TBE
  • 10% (w/v) ammonium persulfate (APS) solution
  • 20% acrylamide solution (see protocol 1, step 10)
  • Amersham Hyperscreen intensifying screen (35 × 43 cm, GE Healthcare Life Sciences)
  • Autoradiography cassette (35 × 43 cm, GE Healthcare Life Sciences)
  • Gel plates (33 × 42 cm; CBS Scientific)
  • ImageQuant software (GE Healthcare)
  • Spacers (0.4 mm thick; CBS Scientific) and combs (16 wells; CBS Scientific)
  • Storm 820 phosphorimager (GE Healthcare)
  • ThermoMixer
  • Sephadex G10 spin column (see protocol 1)
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