Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells.
Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells.
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DOI：
10.1093/nar/gki968
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发表时间：
2005
影响因子：
14.9
通讯作者：
Taylor JS
中科院分区：
文献类型：
--
作者：
Fang H;Yue X;Li X;Taylor JS
我们最近已经证明，用64Cu标记的多肽核酸(PNA)与渗透肽Lys4结合可以在小鼠体内对MCF-7肿瘤进行成像，Lys4识别在这些细胞中表达的唯一过表达和非常丰富的N-ras或N-ras相关基因(UNR MRNA)的上游。在这里，我们描述了如何鉴定和鉴定针对UNR mRNA的高亲和力反义PNA。首先，通过我们改进的逆转录酶随机寡核苷酸文库(RT-ROL)方法和我们开发的一系列反义结合位点分析(SAAbs)方法来定位UNR mRNA上的反义结合位点，该方法与最近报道的另一种方法类似。然后用一种新的基于动态斑点杂交的方法对与反义结合位点互补的寡脱氧核苷酸(ODN)的相对结合亲和力进行定性排名。用一种新的基于动力学的溶液分析方法测定了部分ODN的解离常数，发现最佳结合剂在1M盐中的解离常数为300 pm。用N端CysTyr和C端Lys4序列合成与亲和力最高的ODN对应的PNA。这些杂化PNA的离解常数通过基于dynabad的溶液分析确定为对于最高亲和力的结合剂约为10 pm。
We have recently shown that an MCF-7 tumor can be imaged in a mouse by PET with 64Cu-labeled Peptide nucleic acids (PNAs) tethered to the permeation peptide Lys4 that recognize the uniquely overexpressed and very abundant upstream of N-ras or N-ras related gene (unr mRNA) expressed in these cells. Herein we describe how the high affinity antisense PNAs to the unr mRNA were identified and characterized. First, antisense binding sites on the unr mRNA were mapped by an reverse transcriptase random oligonucleotide library (RT-ROL) method that we have improved, and by a serial analysis of antisense binding sites (SAABS) method that we have developed which is similar to another recently described method. The relative binding affinities of oligodeoxynucleotides (ODNs) complementary to the antisense binding sites were then qualitatively ranked by a new Dynabead-based dot blot assay. Dissociation constants for a subset of the ODNs were determined by a new Dynabead-based solution assay and were found to be 300 pM for the best binders in 1 M salt. PNAs corresponding to the ODNs with the highest affinities were synthesized with an N-terminal CysTyr and C-terminal Lys4 sequence. Dissociation constants of these hybrid PNAs were determined by the Dynabead-based solution assay to be about 10 pM for the highest affinity binders.
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DOI：
10.1073/pnas.90.22.10648
发表时间：
1993-11-15
影响因子：
11.1
作者：
PEFFER, NJ;HANVEY, JC;BABISS, LE
通讯作者：
BABISS, LE
影响因子：
4.7
作者：
Gallazzi, F;Wang, Y;Lewis, MR
通讯作者：
Lewis, MR
影响因子：
3.5
作者：
Ma, ZC;Taylor, JS
通讯作者：
Taylor, JS
影响因子：
1.5
作者：
Rao, PS;Tian, X;Wickstrom, E
通讯作者：
Wickstrom, E
影响因子：
15
作者：
Cai, JF;Li, XX;Taylor, JS
通讯作者：
Taylor, JS