Novel cyanine-AMP conjugates for efficient 5' RNA fluorescent labeling by one-step transcription and replacement of [gamma-32P]ATP in RNA structural investigation.
Two novel fluorescent cyanine-AMP conjugates, F550/570 and F650/670, have been synthesized to serve as transcription initiators under the T7 phi2.5 promoter. Efficient fluorophore labeling of 5' RNA is achieved in a single transcription step by including F550/570 and F650/670 in the transcription solution. The current work makes fluorescently labeled RNA readily available for broad applications in biochemistry, molecular biology, structural biology and biomedicine. In particular, site-specifically fluorophore-labeled large RNAs prepared by the current method may be used to investigate RNA structure, folding and mechanism by various fluorescence techniques. In addition, F550/570 and F650/670 may replace [gamma-32P]ATP to prepare 5' labeled RNA for RNA structural and functional investigation, thereby eliminating the need for the unstable and radio-hazardous [gamma-32P]ATP.
Li
Na
N
Department of Chemistry and Biochemistry, University of Southern Mississippi Hattiesburg MS 39406-5043, USA.
Yu
Changjun
C
Huang
Faqing
F
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D001483:Base Sequence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D007211:Indoles / Q000138:chemical synthesis / Q000737:chemistry*; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D012313:RNA / Q000096:biosynthesis / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D014158:Transcription, Genetic
C497832:F550-570 compound; C497833:F650-670 compound; D005456:Fluorescent Dyes; D007211:Indoles; D010761:Phosphorus Radioisotopes; D000249:Adenosine Monophosphate; D012313:RNA; D000255:Adenosine Triphosphate
RNA. 1999 Oct;5(10):1348-56
10573125
Biochemistry. 2005 Mar 22;44(11):4582-90
15766289
Methods. 2001 Mar;23(3):240-54
11243837
EMBO J. 2001 Apr 17;20(8):2051-61
11296237
Methods. 2001 Sep;25(1):19-30
11558994
Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4284-9
11929999
Biopolymers. 2001-2002;61(3):159-79
11987179
Biopolymers. 2001-2002;61(3):224-42
11987183
RNA. 2002 Oct;8(10):1242-52
12403463
Chem Biol. 2002 Nov;9(11):1227-36
12445773
Nucleic Acids Res. 2003 Feb 1;31(3):e8
12560511
Curr Opin Struct Biol. 2003 Feb;13(1):88-97
12581665
RNA. 2003 Dec;9(12):1562-70
14624011
Proc Natl Acad Sci U S A. 2004 Jan 13;101(2):534-9
14704266
Methods Mol Biol. 2004;252:77-108
15017044
Nucleic Acids Res. 2004;32(9):2707-15
15148358
Proc Natl Acad Sci U S A. 2004 Jul 6;101(27):10066-71
15218105
Cytometry. 1990;11(3):418-30
2340776
Science. 1991 Jan 25;251(4992):401-7
1989074
Biochemistry. 1991 May 14;30(19):4821-30
2029524
Science. 1994 Nov 4;266(5186):785-9
7973630
Bioconjug Chem. 1993 Mar-Apr;4(2):105-11
7873641
Biochemistry. 1997 Apr 22;36(16):4718-30
9125492
Science. 1998 Mar 20;279(5358):1940-3
9506944
EMBO J. 1998 Apr 15;17(8):2378-91
9545249
Methods. 1999 May;18(1):60-70
10208817
Methods Enzymol. 2000;317:409-40
10829293