Makrigiorgos G M, Chakrabarti S, Mahmood A
Joint Center for Radiation Therapy and Department of Radiation Oncology, Harvard Medical School, Boston, MA 02215, USA.
Int J Radiat Biol. 1998 Jul;74(1):99-109. doi: 10.1080/095530098141762.
To test the ability of a new fluorescent reagent to label abasic sites in DNA and to use fluorescent energy transfer as a measure of closely-spaced abasic sites in DNA.
A fluorescein-conjugated hydroxylamine derivative (FARP, 5-(((2-(carbohydrazino)-methyl)thio)acetyl)aminofluorescein, aminooxyacetyl hydrazide) that reacts covalently with open chain aldehydes in DNA has been synthesized. Upon depurination of plasmid DNA and reaction with FARP a stable oxime bond is formed between FARP and the generated open chain aldehydes.
By independently quantitating the generated abasic sites, it is shown that most of the generated abasic sites under acidic conditions become fluorescently labelled. The limit of sensitivity with the fluorometer used is approximately 90 femtomole abasic sites, corresponding to 1 abasic site per 17000 base pairs. DNA can be fluorescently labelled over a wide range of FARP:base pair ratios following different extent of depurination, and fluorescent loadings of 1 FARP:20000 base pairs up to 1 FARP:10 base pairs are demonstrated. The heavily labelled samples display significant fluorescence quenching due to the proximity of abasic sites labelled with FARP, that undergo fluorescence energy transfer. Treatment of heavily labelled DNA samples with nuclease P1 results in an increase in fluorescence due to the release of the fluorescent labels in the solution. The relative increase in fluorescence is a quantitative measure of the proximity of labelled abasic sites. Furthermore it is shown that if only 1% of DNA contains abasic sites generated in close proximity (within 10-20 base pairs or less of each other) the resulting quenching is significant enough to detect, even if the rest of the DNA contains isolated abasic sites.
The present approach constitutes a novel fluorescence-based method to detect abasic sites in nucleic acids and demonstrates the feasibility of detecting the presence of closely-spaced damage sites in DNA via fluorescence energy transfer. The technique also comprises a general and convenient method to fluorescently label nucleic acids without introducing strand breaks as a result of the labelling procedure.
测试一种新型荧光试剂标记DNA中无碱基位点的能力，并利用荧光能量转移来衡量DNA中紧密间隔的无碱基位点。
合成了一种与DNA中的开链醛共价反应的荧光素共轭羟胺衍生物（FARP，5-(((2-(氨基脲基)-甲基)硫代)乙酰基)氨基荧光素，氨氧基乙酰肼）。质粒DNA脱嘌呤并与FARP反应后，FARP与生成的开链醛之间形成稳定的肟键。
通过独立定量生成的无碱基位点，结果表明在酸性条件下生成的大多数无碱基位点都被荧光标记。所用荧光计的灵敏度极限约为90飞摩尔无碱基位点，相当于每17000个碱基对中有1个无碱基位点。在不同程度的脱嘌呤后，DNA可以在很宽的FARP:碱基对比例范围内进行荧光标记，并且展示了从1 FARP:20000碱基对到1 FARP:10碱基对的荧光负载量。由于被FARP标记的无碱基位点靠近而发生荧光能量转移，标记严重的样品显示出显著的荧光猝灭。用核酸酶P1处理标记严重的DNA样品会导致荧光增加，这是由于溶液中荧光标记的释放。荧光的相对增加是标记的无碱基位点接近程度的定量指标。此外，结果表明，如果只有1%的DNA含有紧密相邻（彼此在10 - 20个碱基对或更少范围内）生成的无碱基位点，即使其余的DNA含有孤立的无碱基位点，产生的猝灭也足以被检测到。
本方法构成了一种基于荧光检测核酸中无碱基位点的新方法，并证明了通过荧光能量转移检测DNA中紧密间隔损伤位点存在的可行性。该技术还包括一种通用且便捷的方法，可在不由于标记过程而引入链断裂的情况下对核酸进行荧光标记。
