Synthesis and characterization of a peptide nucleic acid conjugated to a D-peptide analog of insulin-like growth factor 1 for increased cellular uptake
Synthesis and characterization of a peptide nucleic acid conjugated to a D-peptide analog of insulin-like growth factor 1 for increased cellular uptake
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DOI：
10.1021/bc9700650
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发表时间：
1997-07-01
影响因子：
4.7
通讯作者：
Wickstrom, E
中科院分区：
文献类型：
--
作者：
Basu, S;Wickstrom, E
DNA疗法在基因特异性、无毒治疗多种疾病方面显示出巨大的潜力。DNA的脱氧核糖磷酸骨架已经以多种方式被修饰以改善核酸酶稳定性和细胞膜渗透性。最近，一种新的DNA衍生物与酰胺骨架，而不是脱氧核糖磷酸骨架，肽核酸（PNA），已显示出巨大的潜力，作为反义剂。尽管PNA与RNA和DNA的杂交非常强烈且特异性很强，但它们被细胞摄取的能力非常差，限制了它们作为核酸结合剂的潜力。为了改善PNA序列的细胞摄取，将其缀合至胰岛素样生长因子1（IGF 1）的D-氨基酸类似物，其选择性地结合至胰岛素样生长因子1（IGF 1 R）的细胞表面受体。将IGF 1D-肽类似物组装在（4-甲基二苯甲基）胺树脂上，然后将PNA延伸作为肽的延续。缀合物和对照序列用C-14放射性标记或用异硫氰酸荧光素荧光标记。在表达低水平鼠IGF 1 R的鼠BALB/c 3 T3细胞、过表达转染的人IGF 1 R基因的p6细胞和作为阴性对照的不表达IGF 1 R的人Jurkat细胞中研究PNA-肽缀合物、肽中具有两个丙氨酸的对照和不具有肽段的对照PNA的细胞摄取。特异性PNA-肽缀合物显示比对照PNA高得多的摄取，但仅在表达IGF 1 R的细胞中。这种方法可以允许细胞特异性和组织特异性应用的PNA作为基因调节剂在体内。
DNA therapeutics show great potential for gene-specific, nontoxic therapy of a wide variety of diseases. The deoxyribose phosphate backbone of DNA has been modified in a number of ways to improve nuclease stability and cell membrane permeability. Recently, a new DNA derivative with an amide backbone instead of a deoxyribose phosphate backbone, peptide nucleic acid (PNA), has shown tremendous potential as an antisense agent. Although PNAs hybridize very strongly and specifically to RNA and DNA, they are taken up by cells very poorly, limiting their potential as nucleic acid binding agents. To improve cellular uptake of a PNA sequence, it was conjugated to a D-amino acid analog of insulin-like growth factor 1 (IGF1), which binds selectively to the cell surface receptor for insulin-like growth factor 1 (IGF1R). The IGF1 D-peptide analog was assembled on (4-methylbenzhydryl)amine resin, and then the PNA was extended as a continuation of the peptide. The conjugate and control sequences were radiolabeled with C-14 or fluorescently labeled with fluorescein isothiocyanate. Cellular uptake of the PNA-peptide conjugate, a control with two alanines in the peptide, and a control PNA without the peptide segment were studied in murine BALB/c 3T3 cells, which express low levels of murine IGF1R, in p6 cells, which are BALB/c 3T3 cells which overexpress a transfected human IGF1R gene, and in human Jurkat cells, which do not express IGF1R, as a negative control. The specific PNA-peptide conjugate displayed much higher uptake than the control PNA, but only in cells expressing IGF1R. This approach may allow cell-specific and tissue-specific application of PNAs as gene-regulating agents in vivo.