Synthesis of Alkyne‐ and Azide‐Modified Oligonucleotides and Their Cyclization by the CuAAC (Click) Reaction
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- Table of Contents
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Abstract
The Cu(I)?catalyzed alkyne?azide cycloaddition (CuAAC) reaction has been used to synthesize cyclic mini?DNA duplexes. The reaction is carried out on 5??alkyne?3??azide?labeled hairpin loop oligonucleotides and proceeds in high yield under mild conditions in as little as 5 min. The resultant duplexes have very high thermal stability and their CD spectra are characteristic of normal B?DNA. Curr. Protoc. Nucleic Acid Chem. 35:4.33.1?4.33.21. © 2008 by John Wiley & Sons, Inc.
Keywords: Click chemistry; CuAAC reaction; oligonucleotide cyclization; mini?duplex
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Synthesis of Propargylamidohexanyl Phosphoramidite
- Basic Protocol 2: Synthesis of Hexynyl Phosphoramidite
- Basic Protocol 3: Synthesis of Succinimidyl‐4‐Azidobutyrate
- Basic Protocol 4: Oligonucleotide Synthesis, Purification, Analysis, and Cyclization
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
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Basic Protocol 1: Synthesis of Propargylamidohexanyl Phosphoramidite
Materials
Basic Protocol 2: Synthesis of Hexynyl Phosphoramidite
Materials
Basic Protocol 3: Synthesis of Succinimidyl‐4‐Azidobutyrate
Materials
Basic Protocol 4: Oligonucleotide Synthesis, Purification, Analysis, and Cyclization
Materials
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Figure 4.33.1 Synthesis of phosphoramidite monomer S.4 and incorporation at the 5′‐end of an oligonucleotide. View Image -
Figure 4.33.2 Synthesis of phosphoramidite monomer S.6 and incorporation at the 5′‐end of an oligonucleotide. View Image -
Figure 4.33.3 Synthesis of active ester S.8 and labeling of the amino group at the 3′‐end of an oligonucleotide. View Image -
Figure 4.33.4 CuAAC reaction of hairpin oligo H‐2 to give cyclic oligo C‐2. View Image -
Figure 4.33.5 Gel electrophoresis analysis of hairpin loops (H; purified) and the click reaction mixture (C) on a 20% polyacrylamide/7 M urea gel 3 hr at a constant power of 20 W, using 0.09 M TBE buffer, pH 8.0. Oligonucleotide sequences are shown in Table . Reprinted from El‐Sagheer et al. () with permission. View Image -
Figure 4.33.6 Mixed injection of equal quantities of purified hairpin loop (H) and cyclic (C) oligonucleotides on capillary electrophoresis. Cyclization of the hairpin loop to give the corresponding cyclic oligonucleotide was confirmed by injection (0.2 OD/100 µL) of each sample individually followed by mixed injection of both samples. ssDNA 100‐R gel, Tris/borate/7 M urea were used (kit no. 477480) on a Beckman Coulter P/ACE MDQ Capillary Electrophoresis System using 32 Karat software, using the following parameters: UV: 254 nm, injection voltage: 10.0 kV, and separation voltage: 9.0 kV (45.0 min duration). Reprinted from El‐Sagheer et al. () with permission. View Image -
Figure 4.33.7 Reversed‐phase HPLC of the reaction mixture for cyclization of hairpin loops H‐1 and H‐2 to give cyclic C‐1 and C‐2. The x axis is time from start of integration (3 min). The y axis is UV absorbance at 280 nm (C‐1) and 292 nm (C‐2). Reprinted from El‐Sagheer et al. () with permission. View Image -
Figure 4.33.8 Gel electrophoresis of crude reaction mixture of cyclization of hairpin H′‐2 using 2 eq of Cu(I) to give the cyclic oligo C′‐2 with varying reaction times. Lane 1: hairpin H′‐2. Lanes 2 to 5: time course of the reaction, shown at 5, 30, and 120 min, respectively. View Image -
Figure 4.33.9 Gel electrophoresis of crude reaction mixture of cyclization of hairpin H‐2 to give the cyclic oligo C‐2 using varying equivalents of Cu(I). Lane 1: hairpin H′‐2. Lanes 2 to 7: reaction with decreasing amounts of Cu(I), shown at 200, 20, 10, 5, 2.5, and 1.25 eq Cu(I), respectively. View Image -
Figure 4.33.10 Derivatives of UV melting curves of hairpin and cyclic oligonucleotides. Melting curves were measured on Cary 400 Scan UV‐Visible Spectrophotometer (Varian) at 5 to 7 µM oligonucleotide in 10 mM phosphate buffer/200 mM NaCl, pH 7.0, to which increasing amounts of formamide were added. Spectra were recorded at 272 nm. T m values were calculated using Cary Win UV Thermal application Software. Reprinted from El‐Sagheer et al. () with permission. View Image -
Figure 4.33.11 CD spectra of cyclic oligonucleotides. Spectra were measured on a Jasco J‐720 spectropolarimeter at 5.0 µM oligonucleotide in 10 mM phosphate buffer/200 mM NaCl, pH 7.0. Spectra were recorded at 100 nm/min with a response time of 1 sec and a bandwidth of 1 nm. A buffer baseline was subtracted from each spectrum to give zero ellipticity at 320 nm. Reprinted from El‐Sagheer et al. () with permission. View Image
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Literature Cited
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