Synthesis and application of a new 2′,3′-isopropylidene guanosine substituted cap analog

Bioorganic and Medicinal Chemistry Letters

Volumn 18, Issue 17, 2008, Pages 4828-4832

  Kore, Anilkumar R ^a   Shanmugasundaram, Muthian ^a   Vlassov, Alexander V ^a  

^a APPLIED BIOSYSTEMS   (United States)

Author keywords

Cap analog; Capping efficiency; HeLa cells; In vitro transcription; Luciferase activity; mRNA stability; T7 RNA polymerase; Translation efficiency

Indexed keywords

ALKENE DERIVATIVE; GUANOSINE DERIVATIVE; HYBRID PROTEIN; LUCIFERASE; MESSENGER RNA; N 7 METHYLGUANOSINE; RNA; UNCLASSIFIED DRUG;

ARTICLE; CHEMICAL MODIFICATION; CONTROLLED STUDY; ENZYME ACTIVITY; GENETIC TRANSFECTION; HELA CELL; HUMAN; HUMAN CELL; IN VITRO STUDY; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; RNA CAPPING; RNA STABILITY; RNA SYNTHESIS; RNA TRANSLATION; SYNTHESIS;

ANIMALS; DNA-DIRECTED RNA POLYMERASES; GUANOSINE; HELA CELLS; HUMANS; LUCIFERASES; LUCIFERASES, FIREFLY; PROTEIN BIOSYNTHESIS; RNA CAP ANALOGS; STRUCTURE-ACTIVITY RELATIONSHIP; VIRAL PROTEINS;

EID: 49849097489     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2008.07.075     Document Type: Article

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23. -.
24. 32P) ATP, 800 (Ci/mmol). In the control reaction, no-cap analog was added. The transcription reactions were incubated at 37 °C for 2 h, after which the reaction mixtures (10 μl) were then applied to a 20% dPAGE gel. Radiation in the gel bands of interest was quantified by a phosphorimager (GE Healthcare).
25. In vitro transcription of the luciferase mRNA. T7 RNA polymerase transcription was performed by using the MEGAscript kit (Ambion, Inc.). All transcription reactions were performed in a 20 μl final volume at the following final concentrations of components: linearized AmbLuc Poly(A) DNA, (1.0 μg total); 1× reaction buffer; ATP, UTP, and CTP, 7.5 mM each; and 50 U/μl of T7 RNA polymerase. Additionally, GTP was present at 7.5 mM in the no-cap control; and in the reactions with cap analog included GTP was present at 1.5 mM, while the cap analog (standard mCAP 1 or modified cap 8) was present at 6.0 mM. The transcription reactions were incubated at 37 °C for 2 h. In order to hydrolyze the remaining plasmid DNA, 1 μl of turbo DNase was added to the reaction mixture, and further incubated at 37 °C for 15 min. Purifications of the RNA from these transcription reactions were done by using the MEGAclear™ Kit (Ambion, Inc.) as per manufacturer's protocol.
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27. ® Gene Expression Cells-to-CT™ Kit (Applied Biosystems). Luciferase mRNA remaining at each time point was converted to a percent of the RNA present at earliest time point (4 h). The results were plotted as remaining intact luciferase mRNA versus time post-electroporation.
28. Luciferase assay. HeLa cells (60,000/well in 24-well plates) were seeded at least 12 h before transfection in growth medium without antibiotics. A complex of 5′-capped RNA was prepared by mixing 600 ng (2 μl) of RNA, 2.5 μl of TFX-20 (Promega), and 300 μl of serum-free DMEM in polystyrene tubes and incubated for 15 min at room temperature. After the incubation, media from the pre-plated HeLa cells were removed and 200 μl of the complex was added to each well. The plates were incubated for 1 h at 37 °C, and then 1 mL of pre-warmed media with serum was added. The transfected plates were incubated at 37 °C. Cells were harvested and lysed at 5, 10, 15, 20, 25, 30, and 40 h post-transfection. The cells were harvested and lysed by removing the media and adding 100 μl of 1× passive lysis buffer (Promega). The plate was mixed carefully to disrupt the cells and 10 μl of cell lysates from each transfections was mixed with 100 μl of luciferase substrate (Promega) and measured immediately on a luminometer (POLARstar OPTIMA, BMG Labtech) in 96-well plates. The fold induction of luciferase protein data was normalized to the control reaction, that is, the no-cap, mRNA Poly(A) transfection results.