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: N ‐( tert ‐Butoxycarbonyloxy)succinimide (Boc‐Osu; Combi‐Blocks, cat. no. SS‐8341) 1 M Tris·Cl, pH 7.6 (see recipe) Triethylamine trihydrofluoride (TEA·3HF), 37% HF (Thermo Scientific Chemicals, cat. no. 350620250) 1‐Butanol (Fisher Chemical, cat. no. A399‐4) Fisherbrand AccuSpin Micro 17 centrifuge (Fisher Scientific, cat. no. 75002461) –20°C freezer

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

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: NHS‐carbonate‐mediated ligand conjugation to the amino‐functionalized solid support, shown here with Boc‐OSu. NOTE: 2′‐tBDSilyl adenosine (n‐bz) CED phosphoramidite (ChemGenes, cat. no. ANP‐5671), 2′‐tBDSilyl guanosine (n‐ibu) CED phosphoramidite (ChemGenes, cat. no. ANP‐5673), 2′‐tBDSilyl cytidine ( n ‐acetyl) CED phosphoramidite (ChemGenes, cat. no. ANP‐6676), and 2′‐tBDSilyl uridine CED phosphoramidite (ChemGenes, cat. no. ANP‐5674) are required for RNA synthesis.

Article Snippet: N ‐( tert ‐Butoxycarbonyloxy)succinimide (Boc‐Osu; Combi‐Blocks, cat. no. SS‐8341) 1 M Tris·Cl, pH 7.6 (see recipe) Triethylamine trihydrofluoride (TEA·3HF), 37% HF (Thermo Scientific Chemicals, cat. no. 350620250) 1‐Butanol (Fisher Chemical, cat. no. A399‐4) Fisherbrand AccuSpin Micro 17 centrifuge (Fisher Scientific, cat. no. 75002461) –20°C freezer

Techniques: Conjugation Assay

Journal: Research and Practice in Thrombosis and Haemostasis

Article Title: A novel biosensor for measuring plasmin activity

doi: 10.1016/j.rpth.2026.106629

Figure Lengend Snippet: Plasmin generation and clot lysis measured using fluorescence resonance energy quenching–based plasmin sensor (FPS) and Boc-Glu-Lys-Lys-AMC. (A) Plasmin generation profiles and clot lysis profiles ( n = 3) in normal pooled plasma obtained by simultaneously measuring FPS hydrolysis and turbidity are shown. These profiles were measured using 1 μM of the FPS where plasmin generation was initiated using 7.4 nM tissue-type plasminogen activator (tPA). (B) Plasmin generation profiles in normal pooled platelet-poor plasma ( n = 3) measured using 1 μM FPS (dashed line) and 1 mM Boc-Glu-Lys-Lys-AMC (EKK; solid line) are shown. For these experiments, 0.1 (orange), 0.05 (green), and 0.025 (pink) nM tPA were used to initiate plasmin generation in the presence of 0.5 μM antibody against antiplasmin (AP AB). (C) The means with SDs for the peak plasmin, peak time, and endogenous plasmin potential (EPP) for the plasmin generation curves in (B) are shown. For all tested tPA concentrations, peak plasmin and EPP were significantly different between experiments measured using FPS and EKK (∗∗∗∗ P < .0001). (D) Plasmin generation profiles ( n = 3) in buffer with 1 μM fibrinogen and 50 nM Glu-plasminogen measured using 1 μM FPS (dashed line) and 1 mM EKK (solid line) where plasmin generation was initiated using 0.1 (red), 0.05 (blue), and 0.025 (purple) nM tPA are shown. (E) The means with SDs for the peak plasmin, peak time, and EPP for the plasmin generation curves in (D) are shown. For all tested tPA concentrations, peak plasmin and EPP were significantly different between experiments measured using FPS and EKK (∗∗∗∗ P < .0001). (F) Clot lysis profiles ( n = 3) in normal pooled plasma measured with the addition of 1 μM FPS (orange), 1 mM EKK (green), or Tris-buffered saline (black) in the presence of 0.5 μM AP AB are shown. For these experiments, 0.1 (solid line) and 0.025 (dashed line) nM tPA were used to initiate plasmin generation. (G) The half clot lysis times for when 0.1 and 0.025 nM tPA were used to initiate plasmin generation in the presence of 0.5 μM AP AB in (F) are shown. For these experiments, there were no significant differences in half clot lysis times between when buffer and FPS were added to plasma (nonsignificant [ns]; P > .05). However, EKK significantly altered half clot lysis times when compared with buffer (∗∗ P < .01). (H) The means with SDs and coefficient of variations (CVs) for the peak plasmin, peak time, EPP, and half clot lysis times for plasmin generation profiles and clot lysis profiles of 13 individuals ( n = 3 for 12 individuals; n = 2 for 1 individual because of experimental error) when 0.5 nM tPA was used to initiate plasmin generation are shown.

Article Snippet: To determine the specificity of FPS and Boc-Glu-Lys-Lys-AMC for plasmin, 1 μM of FPS or 1 mM of Boc-Glu-Lys-Lys-AMC was incubated with 10 nM plasmin; 100 nM thrombin; activated protein C; factor [F]IXa, FXa, and kallikrein; 50 and 100 nM FXIa for FPS and Boc-Glu-Lys-Lys-AMC, respectively (Enzyme Research Laboratories); and 100 nM tPA or uPA.

Techniques: Lysis, Fluorescence, Clinical Proteomics, Saline