Journal: Current Protocols

Article Title: A Facile Strategy for 3′‐Terminal Functionalization of DNA and RNA via On‐Column Conjugation with Integration into Tandem Oligonucleotide Synthesis

doi: 10.1002/cpz1.70374

Figure Lengend Snippet: ( A ) Synthesis of the crude 5′‐ O ‐(4,4′‐dimethoxytrityl)‐3′‐ O ‐sucinimide thymidine ( 1 ) for use in solid support reactions. ( B ) Activation of R ‐CPG using DSC‐mediated mixed carbonate synthesis. ( C ) On‐solid‐support construction of a putrescine carbamate‐linked nucleoside, which can be used for strand elongation subsequently. Reagent abbreviations: DSC, N,N ′‐disuccinimidyl carbonate, CPR II CPG, chemical phosphorylation reagent II control pore glass. Note : All round‐bottom flasks should be oven‐dried at 110°C for at least 2 hr before DSC activation to ensure complete removal of residual moisture. In addition, 3‐Å or 4‐Å molecular sieves should be preactivated if long‐term storage, defined as 2‐3 days, of the crude carbonate solution is anticipated, as they help maintain anhydrous conditions and minimize hydrolysis of the mixed carbonate. An automated dispensing pipettor should be used for measuring liquid volumes, and a scoopula should be used for transferring solid reagents to ensure accuracy and reproducibility. The following reagents are needed to make a mixed DNA oligonucleotide: 3% (w/v) trichloroacetic acid in dichloromethane (DCM; ChemGenes, cat. no. RN‐1462); cap A (acetic anhydride/pyridine/tetrahydrofuran [THF]; ChemGenes, cat. no. RN‐1458); cap B (10% N ‐methylimidazole in THF; ChemGenes, cat. no. RN‐1481); activation reagent (0.25 M of 5‐ethylthio‐tetrazole in ACN; ChemGenes, cat. no. RN‐1466); oxidation solution (0.02 M iodine/pyridine/H 2 O/THF; ChemGenes, cat. no. RN‐1455); acetonitrile (ACN), DNA synthesis grade (Fisher Scientific, cat. no. A998‐4); 2′‐deoxyadenosine (n‐bz) CED phosphoramidite (ChemGenes, cat. no. ANP‐5551); 2′‐deoxyguanosine (n‐ibu) CED phosphoramidite (ChemGenes, cat. no. ANP‐5553); 2′‐deoxycytidine ( n ‐acetyl) CED phosphoramidite (ChemGenes, cat. no. ANP‐5560); and thymidine CED phosphoramidite (ChemGenes, cat. no. ANP‐5554).

Article Snippet: In a 1‐liter glass bottle, add 100 ml ACN, DNA synthesis grade (Fisher Scientific, cat. no. A998‐4), and 50 ml of 1 M Tris·Cl stock solution, pH 7.6 (see recipe below).

Techniques: Activation Assay, Phospho-proteomics, Control, Transferring, DNA Synthesis