Cellular uptake, distribution, and stability of 10-23 deoxyribozymes
Cellular uptake, distribution, and stability of 10-23 deoxyribozymes
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DOI：
10.1089/108729002761381276
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发表时间：
2002-10-01
期刊：
影响因子：
--
通讯作者：
Sun, LQ
中科院分区：
文献类型：
--
作者：
Dass, CR;Saravolac, EG;Sun, LQ
在几种细胞系中检查了 33 聚体脱氧核酶寡核苷酸 (DNAzyme) 的细胞摄取、细胞内分布和稳定性。 PAGE 分析显示，DNAzyme 与 DOTAP 或 Superfect 转染试剂之间存在微弱关联，电荷比对培养细胞的毒性最小。通过放射性标记的 DNAzyme 的细胞分级分离、FACS 以及通过 FITC 标记和 TAMRA 标记的 DNAzyme 的荧光显微镜分析来分析细胞摄取。改变 DNAzyme 的大小和化学性质并不会显着影响细胞的摄取。通过荧光显微镜检查多聚甲醛固定的细胞表明，DNAzyme 主要分布在细胞核周围的点状结构中，并且在转染开始后 24 小时内未观察到大量递送至细胞核。在人血清或血浆中的孵育表明，与未修饰形式（t(1/2) 大约 70 分钟）相比，3'-倒位修饰大大提高了 DNAzyme 的稳定性（t(1/2) 大约 22 小时）。 3'-倒位修饰的 DNAzyme 在细胞摄取过程中保持稳定，并且可以在转染后 24 小时从细胞中提取具有催化活性的寡核苷酸。在平滑肌细胞增殖测定中，针对c-myc基因的修饰DNAzyme表现出比未修饰版本更强的抑制作用。本研究表明，具有 3'-倒位的 DNAzyme 很容易被递送到培养的细胞中，并且在血清和细胞内功能稳定数小时。
The cellular uptake, intracellular distribution, and stability of 33-mer deoxyribozyme oligonucleotides (DNAzymes) were examined in several cell lines. PAGE analysis revealed that there was a weak association between the DNAzyme and DOTAP or Superfect transfection reagents at charge ratios that were minimally toxic to cultured cells. Cellular uptake was analyzed by cell fractionation of radiolabeled DNAzyme, by FACS, and by fluorescent microscopic analysis of FITC-labeled and TAMRA-labeled DNAzyme. Altering DNAzyme size and chemistry did not significantly affect uptake into cells. Inspection of paraformaldehyde-fixed cells by fluorescence microscopy revealed that DNAzyme was distributed primarily in punctate structures surrounding the nucleus and that substantial delivery to the nucleus was not observed up to 24 hours after initiation of transfection. Incubation in human serum or plasma demonstrated that a 3'-inversion modification greatly increased DNAzyme stability (t(1/2) approximate to 22 hours) in comparison to the unmodified form (t(1/2) approximate to 70 minute). The 3'-inversion-modified DNAzymes remained stable during cellular uptake, and catalytically active oligonucleotide could be extracted from the cells 24 hours posttransfection. In smooth muscle cell proliferation assay, the modified DNAzyme targeting the c-myc gene showed a much stronger inhibitory effect than did the unmodified version. The present study demonstrates that DNAzymes with a 3'-inversion are readily delivered into cultured cells and are functionally stable for several hours in serum and within cells.