Department of Chemistry, Bar-Ilan University, Ramat-Gan, Israel.
Institute of Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel.
Curr Protoc Nucleic Acid Chem. 2020 Mar;80(1):e104. doi: 10.1002/cpnc.104.
Nucleoside intercalator conjugates (NICs) describe an innovative methodology developed in our research group for preparation of fluorescence turn-on DNA hybridization probes targeting specific mRNA sequences (e.g., breast cancer markers). In this methodology, we conjugate a non-fluorescent intercalator to the base of a nucleic acid (e.g., uracil) via a flexible spacer. This modified monomer can be incorporated into oligonucleotides by solid-phase synthesis and a large fluorescence enhancement is observed when the modified oligonucleotide is hybridized with its complementary strand due to intercalation of the fluorophore between the two strands. 5-(6-p-Methoxybenzylidene imidazolinone-1-hexene)-2'-deoxyuridine (dU ) is a synthetic monomer to which 4-methoxybenzylidene imidazolinone (MBI), the fluorescent chromophore of green fluorescent protein (GFP), has been conjugated via a flexible spacer. The detection of human epidermal growth factor receptor 2 (HER2) mRNA by this probe has already been established by our group. The fluorescent intensity of the single-strand DNA can be considered as negligible due to the free rotation of the fluorophore. Upon hybridization, however, the flexible spacer allows for the intercalation of the fluorophore between the hybridized strands, giving rise to enhanced fluorescence and indicating the presence of target mRNA. 3,5-Difluoro-4-methoxybenzylidene (DFMBI) has enhanced photophysical properties compared to MBI fluorophore. This protocol describes a simple, reliable, efficient, and general method for the synthesis of improved derivative dU as a monomer of fluorescent turn-on DNA hybridization probe with application for detection of HER2 mRNA. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 5-[(6)-3,5-difluoro-4-methoxybenzylidene imidazolinone-1-hexene]-2'-deoxyuridine.
核苷嵌入剂缀合物 (NICs) 描述了我们研究小组开发的一种创新方法，用于制备针对特定 mRNA 序列（例如乳腺癌标志物）的荧光开启 DNA 杂交探针。在这种方法中，我们通过柔性间隔物将非荧光嵌入剂缀合到核酸（例如尿嘧啶）的碱基上。这种修饰的单体可以通过固相合成掺入寡核苷酸中，并且当修饰的寡核苷酸与其互补链杂交时，由于荧光团在两条链之间的嵌入，观察到较大的荧光增强。5-(6-p-甲氧基苯亚甲基咪唑啉酮-1-己烯)-2'-脱氧尿苷 (dU) 是一种合成单体，其中通过柔性间隔物将绿色荧光蛋白 (GFP) 的荧光发色团 4-甲氧基苯亚甲基咪唑啉酮 (MBI) 缀合。我们小组已经通过该探针建立了人表皮生长因子受体 2 (HER2) mRNA 的检测。由于荧光团的自由旋转，单链 DNA 的荧光强度可以忽略不计。然而，在杂交后，柔性间隔物允许荧光团在杂交链之间嵌入，从而产生增强的荧光，并表明存在靶 mRNA。与 MBI 荧光团相比，3,5-二氟-4-甲氧基苯亚甲基 (DFMBI) 具有增强的光物理性质。本协议描述了一种简单、可靠、高效和通用的方法，用于合成改进的 dU 衍生物作为荧光开启 DNA 杂交探针的单体，用于 HER2 mRNA 的检测。 © 2020 年 John Wiley & Sons, Inc. 基本方案：5-[(6)-3,5-二氟-4-甲氧基苯亚甲基咪唑啉酮-1-己烯]-2'-脱氧尿苷的合成。
