Chakrabarti S, Mahmood A, Makrigiorgos G M
Joint Center for Radiation Therapy and Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.
Int J Radiat Biol. 1999 Aug;75(8):1055-65. doi: 10.1080/095530099139827.
To test the ability of FARPhC and FARP, two novel fluorescent reagents, to label aldehyde-containing sites (principally abasic sites) generated in DNA by the radiomimetic drug bleomycin, and to use fluorescent energy transfer from FARPhc (donor) to FARP (acceptor) to quantitate such closely-spaced sites.
FARPhc, 7-hydroxycoumarin-3-carboxylic acid (((((amino-oxymethyl) carbonyl) hydrazino) carbonylethyl) amide) was synthesized with a protocol similar to the one recently reported for FARP (a fluorescein-based probe).
Both FARPhc and FARP form stable oxime bonds with the open-chain aldehydes generated upon acidic depurination of DNA. Plasmid DNA exposed to bleomycin-Fe(III)-ascorbate undergoes extensive strand breakage, and upon subsequent reaction with FARPhc and/or FARP it becomes fluorescently labelled, indicating the generation of aldehyde-containing sites. The binding of the probes to calf thymus or plasmid DNA results in significant fluorescent energy transfer among closely-spaced fluorophores, as revealed by the fluorescence increase following digestion of fluorescently labelled samples with nuclease P1. The fluorescent quenching is most evident when both FARPhc and FARP are used simultaneously to trap aldehyde sites. When single-stranded oligonucleotides engineered to contain either one or two closely spaced bleomycin binding sites are exposed to bleomycin and then fluorescently labelled, the oligonucleotides demonstrate significantly increased fluorescent energy transfer with two binding sites indicating a dependence of aldehyde site generation and clustering on the local sequence of a single strand.
A new detection method for DNA damage induced by bleomycin following fluorescent labelling of aldehyde group-containing sites (FLAGS) and their clustering via fluorescent energy transfer is demonstrated. The method is applicable to any form of DNA. This work may lead to a general approach for the quantification of multiply damaged sites in DNA, a subset of DNA lesions that may have major biological significance.
测试两种新型荧光试剂FARPhC和FARP标记由放射模拟药物博来霉素在DNA中产生的含醛位点（主要是无碱基位点）的能力，并利用从FARPhC（供体）到FARP（受体）的荧光能量转移来定量此类紧密间隔的位点。
FARPhC，7-羟基香豆素-3-羧酸（(((氨基-氧甲基)羰基)肼基)羰基乙基)酰胺）的合成方案与最近报道的FARP（一种基于荧光素的探针）的方案相似。
FARPhC和FARP都能与DNA酸性脱嘌呤后产生的开链醛形成稳定的肟键。暴露于博来霉素-Fe(III)-抗坏血酸的质粒DNA会发生广泛的链断裂，随后与FARPhC和/或FARP反应时会被荧光标记，表明产生了含醛位点。探针与小牛胸腺或质粒DNA的结合导致紧密间隔的荧光团之间发生显著的荧光能量转移，这通过用核酸酶P1消化荧光标记样品后荧光增加得以揭示。当同时使用FARPhC和FARP捕获醛位点时，荧光猝灭最为明显。当设计含有一个或两个紧密间隔的博来霉素结合位点的单链寡核苷酸暴露于博来霉素然后进行荧光标记时，寡核苷酸在有两个结合位点时显示出显著增加的荧光能量转移，表明醛位点的产生和聚集依赖于单链的局部序列。
证明了一种新的检测方法，即通过对含醛基团位点进行荧光标记（FLAGS）以及通过荧光能量转移对其聚集情况进行检测，来检测博来霉素诱导的DNA损伤。该方法适用于任何形式的DNA。这项工作可能会导致一种通用方法，用于定量DNA中多重损伤位点，这是一类可能具有重大生物学意义的DNA损伤子集。
