Yang Bo, Jinnouchi Akiko, Usui Kazuteru, Katayama Tsutomu, Fujii Masayuki, Suemune Hiroshi, Aso Mariko
†Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
‡Department of Biological and Environmental Chemistry, School of Humanity-Oriented Science and Engineering, Kinki University, 11-6 Kayanomori, Iizuka, Fukuoka 820-8555, Japan.
Bioconjug Chem. 2015 Aug 19;26(8):1830-8. doi: 10.1021/acs.bioconjchem.5b00361. Epub 2015 Aug 5.
We evaluated the efficacy of bioconjugation of oligodeoxynucleotides (ODNs) containing 1,4-dicarbonyl groups, a C4'-oxidized abasic site (OAS), and a newly designed 2'-methoxy analogue, via reductive amination with lysine residues. Dicarbonyls, aldehyde and ketone at C1- and C4-positions of deoxyribose in the ring-opened form of OAS allowed efficient reaction with amines. Kinetic studies indicated that reductive amination of OAS-containing ODNs with a proximal amine on the complementary strand proceeded 10 times faster than the corresponding reaction of an ODN containing an abasic site with C1-aldehyde. Efficient reductive amination between the DNA-binding domain of Escherichia coli DnaA protein and ODNs carrying OAS in the DnaA-binding sequence proceeded at the lysine residue in proximity to the phosphate group at the 5'-position of the OAS, in contrast to unsuccessful conjugation with abasic site ODNs, even though they have similar aldehydes. Theoretical calculation indicated that the C1-aldehyde of OAS was more accessible to the target lysine than that of the abasic site. These results demonstrate the potential utility of cross-linking strategies that use dicarbonyl-containing ODNs for the study of protein-nucleic acid interactions. Conjugation with a lysine-containing peptide that lacked specific affinity for ODN was also successful, further highlighting the advantages of 1,4-dicarbonyls.
我们评估了通过与赖氨酸残基进行还原胺化反应，对含有1,4 - 二羰基、C4'-氧化无碱基位点（OAS）和新设计的2'-甲氧基类似物的寡脱氧核苷酸（ODN）进行生物共轭的效果。在OAS的开环形式中，脱氧核糖C1和C4位的二羰基、醛和酮能够与胺高效反应。动力学研究表明，含OAS的ODN与互补链上近端胺的还原胺化反应比含C1 - 醛无碱基位点的ODN的相应反应快10倍。大肠杆菌DnaA蛋白的DNA结合结构域与在DnaA结合序列中携带OAS的ODN之间的高效还原胺化反应发生在OAS 5'位磷酸基团附近的赖氨酸残基处，与之形成对比的是，即使无碱基位点ODN具有相似的醛基，但与它们的共轭反应却不成功。理论计算表明，OAS的C1 - 醛比无碱基位点的C1 - 醛更容易接近目标赖氨酸。这些结果证明了使用含二羰基ODN的交联策略在研究蛋白质 - 核酸相互作用方面的潜在效用。与对ODN缺乏特异性亲和力的含赖氨酸肽的共轭反应也取得了成功，进一步突出了1,4 - 二羰基的优势。
