UNLABELLED: An important limitation restricting antisense nuclear medicine imaging is low radioactivity accumulations in target cells. The Tat peptide (Tat), a basic domain of the HIV Tat protein, has been shown to enhance cell accumulation of various biomolecules. PURPOSE: The influence of Tat, as a cationic carrier, on the accumulation in cell culture of anionic antisense DNAs bound electrostatically rather than covalently was investigated. To establish specificity of the accumulation, antisense DNA and control sequence were studied along with four different peptides. The technique of in situ reverse transcription was used to assay the in vivo hybridization of antisense DNA to the target mRNA in cultured live cells when transducted with the Tat peptide. METHODS: Uniform phosphorothioated DNAs were radiolabeled with 99mTc via Hynic/tricine. This 18 mer antisense DNA against RI alpha mRNA along with its sense and random control was studied in ACHN cells with the four peptides as carriers. RESULTS: The addition of Tat significantly increased cell accumulations. At 12 hours accumulations went from 14% to 45% for the antisense DNA and from 4% to 12% for control. Furthermore, an antisense effect was again suggested, now with the Tat carrier, by the significantly higher accumulation of 99mTc on both antisense DNAs vs. controls. Moreover, the accumulated antisense DNA enhanced with the Tat carrier was capable of priming reverse transcription as determined by an in situ assay suggesting that the DNA could escape from entrapment in endosome or lysosome vesicles for hybridization. However, differences in cellular accumulation with either Tat compared to either scrambled peptide were not significant, showing that the Tat in this study was functioning merely as a cationic carrier. CONCLUSIONS: Although electrostatic binding of antisense DNA to Tat is convenient, the association may mask the unique transduction properties of the peptide. Nevertheless, a promising improvement in cellular accumulation of antisense DNA was observed through the use of these carriers. In addition, at least a fraction of the transducted DNA appears to be free of entrapment to hybridize to its mRNA target in live cells.
UNLABELLED: An important limitation restricting antisense nuclear medicine imaging is low radioactivity accumulations in target cells. The Tat peptide (Tat), a basic domain of the HIV Tat protein, has been shown to enhance cell accumulation of various biomolecules. PURPOSE: The influence of Tat, as a cationic carrier, on the accumulation in cell culture of anionic antisense DNAs bound electrostatically rather than covalently was investigated. To establish specificity of the accumulation, antisense DNA and control sequence were studied along with four different peptides. The technique of in situ reverse transcription was used to assay the in vivo hybridization of antisense DNA to the target mRNA in cultured live cells when transducted with the Tat peptide. METHODS: Uniform phosphorothioated DNAs were radiolabeled with 99mTc via Hynic/tricine. This 18 mer antisense DNA against RI alpha mRNA along with its sense and random control was studied in ACHN cells with the four peptides as carriers. RESULTS: The addition of Tat significantly increased cell accumulations. At 12 hours accumulations went from 14% to 45% for the antisense DNA and from 4% to 12% for control. Furthermore, an antisense effect was again suggested, now with the Tat carrier, by the significantly higher accumulation of 99mTc on both antisense DNAs vs. controls. Moreover, the accumulated antisense DNA enhanced with the Tat carrier was capable of priming reverse transcription as determined by an in situ assay suggesting that the DNA could escape from entrapment in endosome or lysosome vesicles for hybridization. However, differences in cellular accumulation with either Tat compared to either scrambled peptide were not significant, showing that the Tat in this study was functioning merely as a cationic carrier. CONCLUSIONS: Although electrostatic binding of antisense DNA to Tat is convenient, the association may mask the unique transduction properties of the peptide. Nevertheless, a promising improvement in cellular accumulation of antisense DNA was observed through the use of these carriers. In addition, at least a fraction of the transducted DNA appears to be free of entrapment to hybridize to its mRNA target in live cells.
Authors: Meiduo Hu; Paul Chen; Judy Wang; Deborah A Scollard; Katherine A Vallis; Raymond M Reilly Journal: Eur J Nucl Med Mol Imaging Date: 2006-09-26 Impact factor: 9.236