DNA脱碱基位点的检测方法及其生物学研究进展

DNA中糖苷键断裂形成的脱碱基位点（脱嘌呤/嘧啶位点,AP位点）是常见的DNA损伤类型之一,由核苷酸自发水解产生,也是DNA碱基切除修复途径中的关键中间体。若修复不及时,可能会导致DNA复制阻滞和DNA链断裂,产生突变和细胞毒性,同时还会引起形成DNA交联或DNA－蛋白质交联的损伤,因此对于AP损伤的检测有助于理解细胞氧化应激损伤和基因毒性物质的毒性评价。目前检测AP位点的方法有14C或32P后标记法、酶联免疫吸附分析（ELISA）法和液相色谱－质谱（LC－MS）技术等。该文重点概述DNA中AP位点的检测方法和生物学研究进展,并展望了AP位点损伤相关研究的前景。     

基于核酸脱碱基位点的铽离子荧光增强型单核苷酸多态性识别研究

基于核酸脱碱基(AP)位点构建了无机配体稀土铽离子(Tb^3＋)荧光增强型单核苷酸多态性(SNP)识别方法. 在目标链靶标碱基对应的探针链上相应位置引入AP位点, 发现Tb^3＋可以选择性地结合在AP位点, 光激发时发生从DNA碱基到结合的Tb^3＋的能量转移, 使Tb^3＋特征荧光显著增强. 这种荧光增强作用与靶标碱基及AP位点侧翼碱基类型密切相关. 当靶标碱基和侧翼碱基为G时, 荧光最强. 该方法可用于区分肿瘤抑制基因p53密码子177位的C/G碱基变异.     

DNA脱碱基位点的检测与应用

脱碱基位点是一种常见的DNA损伤,源于N-糖苷键断裂而使碱基脱落。辐射、烷基化试剂和一些抗癌药物等可能会造成碱基脱落,因此脱碱基位点作为标志性损伤能够帮助疾病早期筛查、药物毒副作用评价、环境污染物毒性评价等。目前已有不同的检测方法用于脱碱基位点的定量、定性分析,包括32P后标记法、LC-MS、ELISA及化学探针检测法等。另一方面,由于脱碱基位点在双链DNA内形成疏水空腔,能够结合小分子,使得脱碱基位点作为结合位点被用于小分子检测、构建适配体传感器及SNP检测。本文简要概述目前为止对DNA脱碱基位点的化学探针检测法研究进展以及含有脱碱基位点DNA的应用研究进展,并展望其发展趋势。     

小分子识别DNA脱碱基位点的电化学研究:选择性机理

在活体细胞DNA中每106个碱基含有数十个到数百个AP位点.这些位点能被AP核酸酶识别并通过BER途径所修复.小分子除抑制DNA损伤响应机制而发挥癌症治疗作用外,如果特性药物能强结合到致癌基因的AP位点上,进而影响基因调控与表达,对正确理解药物小分子在基因水平的影响及抗癌药物开发将是一个非常有吸引力的研究方向.2-氨基-7-甲基-1,8-二氮杂萘(AMND)能选择性地与胞嘧啶结合而用于SNP的检测~([1]),本文研究了AMND在AP位点与上下碱基对的堆叠作用.     

色氨酸标记的GCN4单体肽与DNA位点的分子识别

酵母转录激活因子GCN4调控细胞中氨基酸的生物合成, 是典型的含bZIP结构域的DNA结合蛋白.本文合成了天然蛋白GCN4的碱性区（226-252）, 并在其N末端引入色氨酸残基W, 做为单体肽GCN4-W.圆二色(CD)实验表明, 突变后的单体肽仍能序列特异性识别DNA结合位点AP-1和ATF/CREB.用荧光滴定方法获得了GCN4-W与DNA位点结合形成复合物的表观解离常数.     

基于纳米金比色检测NOS1AP基因单碱基突变

基于单、双链DNA与纳米金颗粒间的不同静电作用, 建立了一种基于颜色反应检测NOS1AP基因单碱基突变的方法. 根据NOS1AP基因的单碱基多态位点设计检测探针、互补靶序列及带有单碱基突变序列寡核苷酸DNA. 室温下, 检测探针分别与互补序列、单碱基突变序列在缓冲液中进行杂交, 再分别加入纳米金溶液以及NaCl溶液. 用肉眼可以观察到纳米金溶液在两种不同杂交溶液中产生明显不同的颜色变化. 这种变化可通过紫外-可见分光光度计测定纳米金溶液的紫外吸收峰值的变化来证实. 实验结果表明, 纳米金溶液在一定浓度NaCl存在的条件下, 对互补双链NOS1AP DNA及单碱基突变NOS1AP DNA呈现出不同的颜色反应及紫外吸收光谱的改变. 此方法可望用于相关疾病的医学诊断及单碱基突变的检测.     

荧光小分子2-氨基-5,6,7-三甲基-1,8-萘啶识别dsDNA中胞嘧啶凸出

双链DNA（dsDNA）中单碱基凸出结构（bulge structure）具有重要生物学意义,这种结构也是DNA靶向药物的目标部位之一.荧光小分子2-氨基-5,6,7-三甲基-1,8-萘啶（ATMND）能够通过氢键识别胞嘧啶（cytosine）,因而对dsDNA中凸出的胞嘧啶表现出明显的特异性结合.与其余三种凸出的碱基相比,ATMND与凸出部位胞嘧啶的结合伴随着ATMND荧光的明显猝灭,因而可以用于胞嘧啶凸出结构的识别.利用解旋温度测量、荧光、圆二色光谱对ATMND和存在胞嘧啶凸出结构的dsDNA相互作用进行了研究.荧光滴定结果表明ATMND和dsDNA中凸出部位未配对的胞嘧啶的结合常数K11=4.8×105M？1.通过对含胞嘧啶凸出结构的dsDNA与ATMND结合前后的解旋温度曲线进行解析,发现胞嘧啶凸出结构相邻碱基对凸出的胞嘧啶与ATMND的结合有较大的影响.荧光测量结果也表明ATMND荧光的猝灭效率与凸出结构相邻碱基的类型有关,当相邻碱基为鸟嘌呤（guanine,G）时,荧光猝灭效率最高.基于dsDNA中凸出的碱基对ATMND荧光猝灭效率存在明显差异这一现象,设计了探针DNA实现了乳腺癌相关基因（PGR gene rs3740753）中单核苷酸多态性（G/C变异）的荧光分型.     

N1-甲基鸟嘌呤阳离子脱质子动力学的研究

在所有DNA碱基中, 鸟嘌呤碱基G具有最低的氧化电位, 导致其最容易被氧化. G碱基被单电子氧化成为G正离子自由基(G^+·), G^+·存在两个脱质子位点, 其中脱嘧啶环上亚氨基质子N¹-H比脱环外氨基质子N²-H更有利, 因而在普通G碱基中研究脱N²-H的过程无法排除脱N¹-H过程的干扰, 使得其脱N²-H的动力学迄今尚不明确. 在本文中, 通过将G碱基上的N¹-H用CH₃取代(即mG), 采用纳秒时间分辨瞬态紫外可见吸收光谱方法研究了mG碱基单电子氧化后脱质子N²-H的动力学. 根据瞬态紫外可见吸收光谱, 确定了mG^+·脱质子的产物是mG(N²-H)^·, 即脱质子的位点是N²-H. 进一步通过测量mG(N²-H)^·的生成速率常数与mG的浓度依赖关系, 得到室温下SO₄^-·单电子氧化mG生成 mG^+·的速率常数为(3.7±0.1)×10⁹ L·mol^-1·s^-1以及 mG^+·脱N²-H的速率常数为(7.1±0.2)×10⁶ s^-1. 并通过检测不同温度下mG^+·脱N²-H的速率常数, 利用阿仑尼乌斯方程得出脱质子N²-H的活化能为19.9±1.0 kJ·mol^-1. 这些结果可为DNA碱基的氧化损伤过程提供更为丰富的动力学信息.     

丁基锡系列化合物与脱氧核糖核酸的相互作用

通过紫外（ultraviolet,UV）光谱和圆二色（circular dichroism,CD）光谱,研究了丁基锡化合物（一丁基锡、二丁基锡和三丁基锡）与脱氧核糖核酸（deoxyribonucleic acid,DNA）的作用方式以及时间和浓度的影响。结果显示丁基锡化合物与DNA的作用是双重的,既作用于DNA的碱基,对双螺旋结构有一定影响,又作用于DNA的磷酸基团,使构象发生变化。但是,丁基锡化合物与脱氧核糖核酸作用的程度和方式与丁基锡种类、时间和浓度等因素有关。一丁基锡倾向于与磷酸基团作用,三丁基锡倾向于与碱基作用,而二丁基锡与两者作用程度相近。短时间内,丁基锡化合物的作用位点通常是DNA的碱基;长时间时,则作用位点往往是DNA的磷酸基团。低浓度的丁基锡化合物倾向与DNA的碱基结合,高浓度的丁基锡化合物倾向与DNA的磷酸基团结合。     

基于金纳米粒子的QCM实时检测DNA错配的研究

利用石英晶体微天平（QCM）技术,用双硫醇分子作为连接剂,将金纳米粒子固定于金电极表面,以人类p53基因片断为DNA探针,研究了其在QCM金电极表面的固定、杂交和错配,重点探讨了金纳米粒子修饰的DNA错配碱基个数和错配位点对杂交的影响。在实验条件下,金纳米粒子在QCM金电极表面的修饰使其灵敏度得到了明显提高;而且,错配碱基个数和错配碱基位点的差异都对杂交产生了不同程度的影响。     

Preferential binding specificity of silver cation to a single nucleobase over base pairs evaluated by abasic site-containing DNA

The binding specificity of silver cations to abasic (AP) site-containing DNA was electrochemically investigated by comparison with the fully matched DNA without the AP site. AP site-containing DNA is designed in a way that only the nucleotide opposite the AP site is variable to allow for coexistence of an unpaired nucleotide and a number of DNA base pairs. The surface of a gold electrode was modified by AP site-containing DNA duplex on which Ag⁺ binding specificity was evaluated. Electrochemical investigations on the AP-DNA-modified electrodes reveal that Ag⁺ preferentially associates to the unpaired nucleotides instead of the coexisted base pairs and shows sequence-dependant binding, especially stronger for purines than for pyrimidines. Additionally, the hydrogen bond pattern moieties of the unpaired nucleotides should be involved in Ag⁺ binding evidenced by a decrease of the redox signal when introducing a ligand with its hydrogen bond moiety complementary to the nucleotide deoxycytidine. This is the first attempt to make a comparison in one DNA molecule for metal ion binding to coexisted unpaired nucleotide and DNA base pairs. The present method demonstrates an easy way for investigating binding specificity of heavy metal ions to AP site in the presence of coexisted DNA base pairs.     

Competitive Assay for Theophylline Based on an Abasic Site‐Containing DNA Duplex Aptamer and a Fluorescent Ligand

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site‐containing DNA duplex aptamer (AP aptamer) in combination with an abasic site‐binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23‐mer AP aptamer (5′‐TCT GCG TCC AGX GCA ACG CAC AC‐3′/5′‐GTG TGC GTT GCC CTG GAC GCA GA‐3′; X : the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM . Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.     

Rational Design for Building Blocks of DNA‐Based Conductive Nanowires through Multi‐Copper Incorporation into Mismatched Base Pairs

Metal‐modified DNA base pairs, which possess potential electrical conductivity and can serve as conductive nanomaterials, have recently attracted much attention. Inspired by our recent finding that multicopper incorporation into natural DNA base pairs could improve the electronic properties of base pairs, herein, we designed two novel multi‐copper‐mediated mismatched base pairs (G_3CuT and A_2CuC), and examined their structural and electronic properties by means of density functional theory calculations. The results reveal that these multi‐Cu‐mediated mismatched base pairs still have planar geometries that are thermodynamically favorable to stability, and their binding energies are close to those of multi‐Cu‐mediated normal base pairs (G_3CuC and A_2CuT). Their HOMO–LUMO gaps and ionization potentials decrease significantly compared to the corresponding natural base pairs. As evidenced by the charge transfer excitation transitions, transverse electronic communication of G_3CuT and A_2CuC is remarkably enhanced, suggesting that they facilitate electron migration along the DNA wires upon incorporation. Further examinations also clarify the possibility to build promising DNA helices using the G_3CuT and/or A_2CuC base pairs. The calculated electronic properties of the three‐layer‐stacked multi‐Cu‐mediated mismatched base pairs illustrate that the Cu_m‐DNA have better conductivity. This work provides perspectives for the development and application of DNA nanowires.     

Toward an Understanding of the Chemical Etiology for DNA Minor‐Groove Recognition by Polyamides

Crescent‐shaped polyamides composed of aromatic amino acids, i.e., 1‐methyl‐1H‐imidazole Im , 1‐methyl‐1H‐pyrrole Py , and 3‐hydroxy‐1H‐pyrrole Hp , bind in the minor groove of DNA as 2 : 1 and 1 : 1 ligand/DNA complexes. DNA‐Sequence specificity can be attributed to shape‐selective recognition and the unique corners or pairs of corners presented by each heterocycle(s) to the edges of the base pairs on the floor of the minor groove. Here we examine the relationship between heterocycle structure and DNA‐sequence specificity for a family of five‐membered aromatic amino acids. By means of quantitative DNase‐I footprinting, the recognition behavior of polyamides containing eight different aromatic amino acids, i.e., 1‐methyl‐1H‐pyrazole Pz , 1H‐pyrrole Nh , 5‐methylthiazole Nt , 4‐methylthiazole Th , 3‐methylthiophene Tn , thiophene Tp , 3‐hydroxythiophene Ht , and furan Fr , were compared with the polyamides containing the parent‐ring amino acids Py, Im , and Hp for their ability to discriminate between the four Watson? Crick base pairs in the DNA minor groove. Analysis of the data and molecular modeling showed that the geometry inherent to each heterocycle plays a significant role in the ability of polyamides to differentiate between DNA sequences. Binding appears sensitive to changes in curvature complementarity between the polyamide and DNA. The Tn / Py pair affords a modest 3‐fold discrimination of T?A vs. A?T and suggests that an S‐atom in the thiophene ring prefers to lie opposite T not A.     

Bulged Guanine is Uniquely Sensitive to Damage Caused by Visible‐Light Irradiation of Ethidium Bound to DNA: A Possible Role in Mutagenesis

The interaction of ethidium bromide (=3,8‐diamino‐5‐ethyl‐6‐phenylphenanthridinium bromide; EB) with a series of duplex DNA oligomers having single‐base bulges and single‐base mis‐pairs was investigated (Fig. 1). Physical and spectroscopic analysis reveals no definitive evidence for selective binding of EB at the bulge or mis‐pair. However, irradiation of the bound EB with VIS light leads to lesions in the DNA selectively in the sequence having a bulged guanine. This reaction is attributed to the formation of an exciplex between the lowest excited singlet state of the EB and the bulged guanine. The exciplex is trapped by H₂O, which initiates a sequence of reactions that lead to piperidine‐requiring strand cleavage at this site. Significantly, the damaged bulged guanine is not recognized by E. coli formamidopyrimidine glycosylase (Fpg), which is part of a base‐excision repair system for oxidative damage to DNA. Thus, DNA containing a bulged guanine and having a bound intercalator may be irreparably damaged by exposure to VIS light, even though normal duplex DNA is relatively inert under these conditions.     

Solution Structure,Mechanism of Replication,and Optimization of an Unnatural Base Pair

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge‐on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivatives of the nucleotide analogue d MMO2 , which forms an unnatural base pair with the nucleotide analogue d 5SICS . Replacing the para‐methyl substituent of d MMO2 with an annulated furan ring (yielding d FMO ) has a dramatically negative effect on replication, while replacing it with a methoxy (d DMO ) or with a thiomethyl group (d TMO ) improves replication in both steady‐state assays and during PCR amplification. Thus, d TMO –d 5SICS , and especially d DMO –d 5SICS , represent significant progress toward the expansion of the genetic alphabet. To elucidate the structure–activity relationships governing unnatural base pair replication, we determined the solution structure of duplex DNA containing the parental d MMO2 –d 5SICS pair, and also used this structure to generate models of the derivative base pairs. The results strongly support the intercalative mechanism of replication, reveal a surprisingly high level of specificity that may be achieved by optimizing packing interactions, and should prove invaluable for the further optimization of the unnatural base pair.     

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe.     

An Isosteric and Fluorescent DNA Base Pair Consisting of 4‐aminophthalimide and 2,4‐diaminopyrimidine as C‐Nucleosides

A 13mer DNA duplex containing the artificial 4‐aminophthalimide:2,4‐diaminopyrimidine (4AP:DAP) base pair in the central position was characterized by optical and NMR spectroscopy. The fluorescence of 4AP in the duplex has a large Stokes shift of Δλ =124 nm and a quantum yield of Φ _F=24 %. The NMR structure shows that two interstrand hydrogen bonds are formed and confirms the artificial base pairing. In contrast, the 4‐N ,N ‐dimethylaminophthalimide moiety prefers the syn conformation in DNA. The fluorescence intensity of this chromophore in DNA is very low and the NMR structure shows no significant interaction with DAP. Primer‐extension experiments with DNA polymerases showed that not only is the 4AP C nucleotide incorporated at the desired position opposite DAP in the template, but also that the polymerase is able to progress past this position to give the full‐length product. The observed selectivity supports the NMR results.     

The Thiophene “Sigma‐Hole” as a Concept for Preorganized,Specific Recognition of G⋅C Base Pairs in the DNA Minor Groove

In spite of its importance in cell function, targeting DNA is under‐represented in the design of small molecules. A barrier to progress in this area is the lack of a variety of modules that recognize G ? C base pairs (bp) in DNA sequences. To overcome this barrier, an entirely new design concept for modules that can bind to mixed G ? C and A ? T sequences of DNA is reported herein. Because of their successes in biological applications, minor‐groove‐binding heterocyclic cations were selected as the platform for design. Binding to A ? T sequences requires hydrogen‐bond donors whereas recognition of the G‐NH₂ requires an acceptor. The concept that we report herein uses pre‐organized N‐methylbenzimidazole (N‐MeBI) thiophene modules for selective binding with mixed bp DNA sequences. The interaction between the thiophene sigma hole (positive electrostatic potential) and the electron‐donor nitrogen of N‐MeBI preorganizes the conformation for accepting an hydrogen bond from G‐NH₂. The compound–DNA interactions were evaluated with a powerful array of biophysical methods and the results show that N‐MeBI‐thiophene monomer compounds can strongly and selectively recognize single G ? C bp sequences. Replacing the thiophene with other moieties significantly reduces binding affinity and specificity, as predicted by the design concept. These results show that the use of molecular features, such as sigma‐holes, can lead to new approaches for small molecules in biomolecular interactions.