Optical antisense tumor targeting in vivo with an improved fluorescent DNA duplex probe.
Fluorescent conjugated DNA oligonucleotides for antisense targeting of mRNA has the potential of improving tumor/normal tissue ratios over that achievable by nuclear antisense imaging. By conjugating the Cy5.5 emitter to the 3' equivalent end of a 25 mer phosphorothioate (PS) antisense major DNA and hybridizing with a shorter 18 mer phosphodiester (PO) complementary minor DNA (cDNA) with the Black Hole inhibitor BHQ3 on its 5' end (i.e., PS DNA25-Cy5.5/PO cDNA18-BHQ3), we previously achieved antisense optical imaging in mice as a proof of this concept. In a process of optimization, we have now evaluated the stability of a small series of duplexes with variable-length minor strands. From these results, a new study anti-mdr1 antisense duplex was selected with a 10 mer minor strand (i.e., PS DNA25-Cy5.5/PO cDNA10-BHQ3). The new study duplex shows stability in serum environments at 37 degrees C and provides a dramatically enhanced fluorescence in KB-G2 (pgp++) cells when compared with KB-31 (pgp+/-) as evidence of antisense dissociation at its mdr1 mRNA target. The duplex was also administered to KB-G2 tumor bearing mice, and when compared to the duplex used previously, the fluorescence from the tumor thigh was more obvious and the tumor-to-background fluorescence ratio was improved. In conclusion, by a process designed to optimize the duplex for optical antisense tumor targeting, the fluorescence signal was improved both in cells and in tumored mice.
Liang
Minmin
M
Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.
Liu
Xinrong
X
Cheng
Dengfeng
D
Nakamura
Kayoko
K
Wang
Yi
Y
Dou
Shuping
S
Liu
Guozheng
G
Rusckowski
Mary
M
Hnatowich
Donald J
DJ
D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles / Q000378:metabolism; D045744:Cell Line, Tumor; D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016373:DNA, Antisense / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D006801:Humans; D008297:Male; D051379:Mice; D009369:Neoplasms / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D011804:Quinolines / Q000378:metabolism
D052160:Benzothiazoles; D015342:DNA Probes; D016373:DNA, Antisense; D011804:Quinolines; C051000:thiazole orange