Johnson Neil P, Baase Walter A, Von Hippel Peter H
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229, USA.
Proc Natl Acad Sci U S A. 2004 Mar 9;101(10):3426-31. doi: 10.1073/pnas.0400591101. Epub 2004 Mar 1.
Circular dichroism is commonly used to investigate the conformations of nucleic acids. However, many biochemical processes implicate conformational changes of particular nucleotide residues within DNA or RNA that cannot be studied by this method, because the CD of these residues is buried in the total signal of the polynucleotide. Here, we report a method to study local conformations of DNA or RNA that is based on the use of the CD of 2-aminopurine (AP) residues as a probe. AP is readily incorporated into DNA in place of adenine and does not significantly alter DNA structure. Unlike adenine, AP is fluorescent and this property has been used for many years to investigate local nucleic acid structure. We show here that the CD spectrum of AP dinucleotide, (AP)(2), exhibits a positive CD band at 326 nm, a spectral region in which nucleic acids (and proteins) do not absorb. Our results show that the bases of (AP)(2) are stacked in a right-handed helical conformation. A low-energy CD band is also observed when this nucleotide dimer is incorporated into double-stranded DNA. Control experiments show that this signal comes from the stacking of adjacent AP residues. We have used this CD signal to provide information about the conformation of the AP dinucleotide at a defined position within single- and double-stranded nucleic acids.
圆二色性常用于研究核酸的构象。然而，许多生化过程涉及DNA或RNA中特定核苷酸残基的构象变化，而这种方法无法对其进行研究，因为这些残基的圆二色性信号被多核苷酸的总信号所掩盖。在此，我们报告一种基于使用2-氨基嘌呤（AP）残基的圆二色性作为探针来研究DNA或RNA局部构象的方法。AP很容易取代腺嘌呤掺入DNA中，并且不会显著改变DNA结构。与腺嘌呤不同，AP具有荧光性，多年来这一特性一直被用于研究局部核酸结构。我们在此表明，AP二核苷酸（AP）₂的圆二色光谱在326 nm处呈现一个正的圆二色带，这是核酸（和蛋白质）不吸收的光谱区域。我们的结果表明，（AP）₂的碱基以右手螺旋构象堆积。当这种核苷酸二聚体掺入双链DNA中时，还会观察到一个低能量的圆二色带。对照实验表明，该信号来自相邻AP残基的堆积。我们利用这种圆二色信号来提供有关单链和双链核酸中特定位置的AP二核苷酸构象的信息。
