噻唑橙核酸荧光探针的合成及光谱性质研究

本文合成了一类可用于核酸分子检测的噻唑橙类(thiazole orange,TO)菁染料4a、4b和5,并对染料结构进行了表征。其中染料4a未见文献报道。三种染料在Tris-HCl(pH=7.0)缓冲溶液中的最大吸收光谱值分别在504nm、502nm、507nm处。荧光发射光谱表明:染料在Tris-HCl(pH=7.0)缓冲溶液中无荧光,加入ds-DNA后荧光显著增强,荧光增强与加入ds-DNA的量之间呈现良好的线性关系。改变染料取代基后得到的染料4a对ds-DNA表现出更优异的荧光增强性能。     

吲哚七甲川类荧光探针在生物体应用的研究现状

菁染料是一种商品化的近红外荧光染料, 其光谱范围位于近红外区域, 此光谱区域内生物基体光吸收或荧光强度较小, 因此利用菁染料对生物体进行成像, 可以降低背景干扰. 吲哚七甲川菁染料是一类具有代表性的菁染料, 其由吲哚杂环、七甲川链和N-取代基侧链组成, 由于具有溶解性好, 最大吸收波长可调, 摩尔消光系数大等优良的光学性质, 被广泛用于肿瘤靶向治疗、蛋白标记、痕量金属离子检测等生物方面. 以吲哚七甲川为母体, 通过向母体中引入活性基团或改变它们的结构, 使探针具有不同的功能, 这已成为生物领域荧光成像的研究热点. 分别从检测金属阳离子、pH变化、小分子和靶向标记肿瘤细胞、蛋白质等方面综述了近年来用于生物体荧光成像的吲哚七甲川类荧光探针的研究进展, 其中也对荧光探针在生物体内(模型)分布、光学成像及代谢方面进行了介绍, 最后讨论了这类荧光探针存在的问题及发展趋势.     

新型不对称五甲川菁染料的合成及光稳定性能研究

合成并表征了5种不对称五甲川菁染料,染料在甲醇中的最大吸收和荧光光谱在646—666nm之间.光降解实验证明两端取代基结构呈不对称的染料,其光稳定性明显高于两端取代基结构对称的染料.染料荧光光谱和pH值的关系表明,染料中引入苯环取代基可以增强染料在酸性或碱性溶液中的稳定性.     

新型水溶性不对称五甲川菁染料的合成、光稳定性及蛋白质的荧光标记

合成并表征了系列水溶性五甲川菁染料, 研究了其在不同溶剂中的光谱性能. 结果表明, 染料在水中的最大吸收和荧光光谱在647~665 nm波长范围内, 荧光量子产率达到0.1左右. 考察了N位取代基对染料水溶液光稳定性的影响, 结果表明, 在N原子上引入带有苯环结构和大体积的磺酸基, 可以提高染料的光稳定性. 高效液相色谱(HPLC)分析结果表明, 染料4a的N-羟基琥珀酰亚胺(NHS)活性酯标记牛血清白蛋白(BSA)的检测限为1.2×10^-8 mol/L, 与紫外检测相比, 检测灵敏度提高了近2个数量级.     

荧光标记染料

荧光标记染料在生命科学领域具有极大的应用前景，基因芯片的研究开发使荧光染料应用于药理研究、药物毒性、药物靶标研究、医学诊断等领域。本文对荧光素、若丹明、菁染料等核酸及蛋白质分析检测用荧光标记染料的结构、性能及其应用特点作了较为详细的介绍。     

新型不对称苯并噻唑三次甲基菁染料的合成及光稳定性研究

合成了系列杂环氮原子具有不同取代基的不对称苯并噻唑三次甲基菁染料,染料结构经过质谱、1H NMR表征.测试了染料在乙醇中的吸收和荧光发射光谱,染料的最大吸收值和荧光发射值分别在628～631和662～666 nm之间.染料在溶剂中几乎无荧光,可以极大的降低染料自身的荧光背景干扰.染料的光降解实验表明:喹啉环上辛烷基取代和噻唑环上苄基取代时,染料的光稳定性最强.染料8a～8d的光降解速率常数分别为6.38×10-4,12.5×10-4,2.68×10-4和6.30×10-4mol min-1.循环伏安实验法测试了染料的氧化电位.     

基于喹啉与苯胺衍生物不对称方酸菁染料的合成与光谱性质

设计合成了四个基于喹啉与苯胺衍生物的不对称方酸菁染料7a～7d,利用1HNMR,MS和元素分析对结构进行了表征.对中间体碘盐3a～3c的合成条件进行了探索,发现随着喹啉6位取代基吸电子能力的增强,亲核取代反应的活性降低,因此需要较为苛刻的条件.对不对称方酸菁的合成方法进行了系统研究,发现不对称方酸菁前体的接入方式是反应成败的关键,并对该不对称方酸菁的吸收光谱与光稳定性进行了系统研究.研究表明,染料的吸收半峰宽较宽,最大吸收随着溶剂极性的增加发生蓝移,表现为负溶剂化效应.光稳定性实验表明,染料的光稳定较好,且喹啉半体6位取代基吸电子能力的增加有利于染料光稳定性的增加.此外,苯胺半体氮上烷基链的长度对染料的光稳定性也有影响.     

“一步法”合成新型不对称三甲川吲哚菁荧光染料及荧光标记

首次合成了一种含非离子水溶性基团的水溶性不对称三甲川吲哚菁荧光染料.以N-(3,5-二(2-(2-甲氧乙氧基)乙氧基)乙氧基)苄基-2,3,3-三甲基-5-磺酸基-3H-吲哚、N-对羧苄基-2,3,3-三甲基-5-磺酸基-3H-吲哚和缩合剂N,N′-二苯基二甲脒为原料,采用"一步法"合成,用自制简易C18反相硅胶填料柱分离即可得到纯品,目标染料经HRMS、NMR表征;测试了染料的紫外光谱性能、荧光光谱性能和光稳定性能;研究了染料标记牛血清白蛋白(BSA)和细胞染色.结果表明:在吲哚"N"原子上引入具有PEG链的非离子水溶性基团,使目标染料合成简便,纯化简单,产率达到73%;荧光量子产率(Φ)达到0.3;标记蛋白质标示率(D/P)为1.87;染料对固定细胞和活细胞染色结果有显著差异,能有效区分细胞存活状态.     

菁染料薄膜的光谱性能及稳定性的研究

本文研究了6种长链菁染料薄膜的光谱特性与光稳定性,以及两种抗氧剂对其薄膜光稳定性的影响。研究表明,结构相近的菁染料利用旋涂法成膜后,光稳定性与其母核的结构有关,依吲哚>喹啉> 唑>噻唑>硒唑顺序而变,与溶液状态下相类似,两种抗氧剂均为有效的单重态氧猝灭剂,都能提高菁染料的光稳定性。且抗氧剂的用量对染料的稳定性也会产生一定的影响     

吲哚碳菁染料的合成及光谱特征

在吡啶中, 吲哚啉碘盐与原乙酸三乙酯缩合合成了一些新的吲哚碳菁染料.根据紫外-可见光谱, 荧光光谱讨论了它们的取代基和光谱特征的关系.     

ECHO probes: a concept of fluorescence control for practical nucleic acid sensing

An excitonic interaction caused by the H-aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence-controlled nucleic acid sensing. This critical review points out the recent advances in exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes, which have a fluorescence-labeled nucleotide in which two molecules of thiazole orange or its derivatives are linked covalently. ECHO probes show absorption shift and emission switching depending on hybridization with the target nucleic acid. The hybridization-sensitive fluorescence emission of ECHO probes and the further modification of probes have made possible a variety of practical applications, such as multicolor RNA imaging in living cells and facile detection of gene polymorphism (144 references).     

A theoretical and experimental study of two thiazole orange derivatives with single- and double-stranded oligonucleotides, polydeoxyribonucleotides and DNA

The effect of interaction with DNA and oligonucleotides on the photophysical properties of two thiazole orange (TO) derivatives, with different side chains (-(CH2)3-N+(CH3)3 and -(CH2)6-I)) linked to the nitrogen of the quinoline ring of the thiazole orange, is presented here. The first one called TO-PRO1 is a commercially available dye, whereas the second one called TO-MET has been specially synthesized for further covalent binding to oligonucleotides with the aim of being used for specific in situ detection of biomolecular interactions. Both photophysical measurements and molecular calculations have been done to assess their possible mode of interaction with DNA. When dissolved in buffered aqueous solutions both derivatives exhibit very low fluorescence quantum yields of 8 x 10(-5) and 2 x 10(-4), respectively. However, upon binding to double-stranded DNA, large spectroscopic changes result and the quantum yield of fluorescence is enhanced by four orders of magnitude, reaching values up to phi F = 0.2 and 0.3, respectively, as a result of an intercalation mechanism between DNA base pairs. A modulation of the quantum yield is observed as a function of the base sequence. The two derivatives also bind with single-stranded oligonucleotides, but the fluorescence quantum yield is not so great as that when bound to double-stranded samples. Typical fluorescence quantum yields of 7 x 10(-3) to 3 x 10(-2) are observed when the dyes interact with short oligonucleotides, whereas the fluorescence quantum yield remains below 10(-2) when interacting with single-stranded oligonucleotides. This slight but significant quantum-yield increase is interpreted as a folding of the single strand around the dye, which reduces the internal rotation of the two heterocycles around the central methine bridge that links the two moieties of the dye. From these properties, it is proposed to link monomer covalently to oligonucleotides for the subsequent detection of target sequences within cells.     

Spectroscopic Characterization of Thiazole Orange-3 DNA Interaction

The interaction of a new derivative of thiazole orange (TO-3) with calf thymus DNA (ctDNA) has been investigated by fluorescence and absorption spectroscopy. When TO-3 binds to ctDNA, absorption bands exhibit significant hypochromicity at low base pair/dye ratio (BP/D ratio), and high BP/D show hyperchromicity with red shift. The spectral changes are attributed to the different species formed between TO-3 and ctDNA in the titration course of the dye molecule with DNA. Multivariate curve resolutions–alternating least squares (MCR–ALS) is applied to the absorption measurements recorded to recover the concentration profiles and the pure spectra of the DNA/TO-3 complexes involved in the process. The binding constant and size of the binding site have been determined spectrophotometrically using the McGhee von Hippel equation. MCR–ALS has been used to reveal the precise concentration profiles of all detectable species formed between ctDNA and TO-3 and their pure spectral profiles.     

Fluorescent properties of oligonucleotide-conjugated thiazole orange probes

The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophilic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2'-deoxyriboadenylate (dA15), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange-oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (phifl) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5'[CACT15CAC3']); (phifl values of between 0.06-0.35 were determined for the double-stranded conjugates. The phifl value (0.17) of thiazole orange, 1-(N,N'-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1: dA15.d5'[CACT15CAC3'] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady-state and time-resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time-resolved studies on the four double-stranded TO-probes revealed that the fluorescent oligonucleotide-thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO-probes in which the isomeric structure of the phosphate-chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher phifl values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl chain to the internucleotidic phosphate (Rp isomer) has the highest phifl and the greatest fraction of the longest-lived fluorescent thiazole orange species (in the hybrid form).     

Excitonic interaction: another photophysical process for fluorescence‐controlled nucleic acid sensing

An excitonic interaction caused by the H‐aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence‐controlled nucleic acid sensing. We designed a fluorescence‐labeled nucleotide in which two thiazole orange dyes were linked covalently. A DNA strand containing this fluorescence‐labeled nucleotide showed absorption at 480 nm before hybridization, whereas an absorption band at 510 nm became predominant when the DNA was hybridized with the complementary strand. The shift in the absorption bands shows the existence of an excitonic interaction between dyes in the nucleotide, and as a result, emission from the doubly thiazole orange‐labeled DNA was well controlled. This clear change in fluorescence intensity depending on hybridization is applicable to multicolor RNA imaging in living cells. © 2010 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 10: 188–196; 2010: Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.201000003     

Influence of Alkyl Chains of Modified Polysuccinimide‐Based Polycationic Polymers on Polyplex Formation and Transfection

The development of polymers with low toxicity and efficient gene delivery remains a significant barrier of nonviral gene therapy. Modification and tuning of chemical structures of carriers is an attractive strategy for efficient nucleic acid delivery. Here, polyplexes consisting of plasmid DNA (pDNA) and dodecylated or non‐dodecylated polysuccinimide (PSI)‐based polycations are designed, and their transfection ability into HeLa cells is investigated by green fluorescent protein (GFP) expressing cells quantification. All cationic polymers show lower cytotoxicity than those of branched polyethyleneimine (bPEI). PSI and bPEI‐based polyplexes have comparable physicochemical properties such as size and charge. Interestingly, a strong interaction between dodecylated polycations and pDNA caused by the hydrophobic moiety is observed in dodecylated PSI derivatives. Moreover, the decrease of GFP expression is associated with lower dissociation of pDNA from polyplexes according to the heparin displacement assay. Besides, a hydrophobization of PSI cationic derivatives with dodecyl side chains can modulate the integrity of polyplexes by hydrophobic interactions, increasing the binding between the polymer and the DNA. These results provide useful information for designing polyplexes with lower toxicity and greater stability and transfection performance.     

Key Structural Elements of Unsymmetrical Cyanine Dyes for Highly Sensitive Fluorescence Turn‐On DNA Probes

Unsymmetrical cyanine dyes, such as thiazole orange, are useful for the detection of nucleic acids with fluorescence because they dramatically enhance the fluorescence upon binding to nucleic acids. Herein, we synthesized a series of unsymmetrical cyanine dyes and evaluated their fluorescence properties. A systematic structure–property relationship study has revealed that the dialkylamino group at the 2‐position of quinoline in a series of unsymmetrical cyanine dyes plays a critical role in the fluorescence enhancement. Four newly designed unsymmetrical cyanine dyes showed negligible intrinsic fluorescence in the free state and strong fluorescence upon binding to double‐stranded DNA (dsDNA) with a quantum yield of 0.53 to 0.90, which is 2 to 3 times higher than previous unsymmetrical cyanine dyes. A detailed analysis of the fluorescence lifetime revealed that the dialkylamino group at the 2‐position of quinoline suppressed nonradiative decay in favor of increased fluorescence quantum yield. Moreover, these newly developed dyes were able to stain the nucleus specifically in fixed HeLa cells examined by using a confocal laser‐scanning microscope.     

Reversed Assembly of Dyes in an RNA Duplex Compared with Those in DNA

We prepared reversed dye clusters by hybridizing two RNA oligomers, each of which tethered dyes (Methyl Red, 4′‐methylthioazobenzene, and thiazole orange) on D ‐threoninols (threoninol nucleotides) at the center of their strands. NMR spectroscopic analyses revealed that two dyes from each strand were axially stacked in an antiparallel manner to each other in the duplex, and were located adjacent to the 3′‐side of a natural nucleobase. Interestingly, this positional relationship of the dyes was completely the opposite of that assembled in DNA that we reported previously: dyes in DNA were located adjacent to the 5′‐side of a natural nucleobase. This observation was also consistent with the circular dichroism of dimerized dyes in which the Cotton effect of the dyes (i.e., the winding properties of two dyes) was inverted in RNA relative to that in DNA. Further spectroscopic analyses revealed that clustering of the dyes on RNA duplexes induced distinct hypsochromicity and narrowing of the band, thus demonstrating that the dyes were axially stacked (i.e., H‐aggregates) even on an A‐type helix. On the basis of these results, we also prepared heterodimers of a fluorophore (thiazole orange) and quencher (Methyl Red) in an RNA duplex. Fluorescence from thiazole orange was found to be strongly quenched by Methyl Red due to the excitonic interaction, so that the ratio of fluorescent intensities of the RNA–thiazole orange conjugate with and without its complementary strand carrying a quencher became as high as 27. We believe that these RNA–dye conjugates are potentially useful probes for real‐time monitoring of RNA interference (RNAi) mechanisms.     

Detection of terminal mismatches on DNA duplexes with fluorescent oligonucleotides

This paper describes the design of terminal-mismatch discriminating fluorescent oligonucleotides (TMDFOs). The method is based on the use of sets of oligo-2'-deoxyribonucleotide probes linked via their 5'-ends, and varying-sized flexible polymethylene chains, to thiazole orange, with the linker being attached to the benzothiazole moiety. The sequence of each set of labelled probes was identical and complementary to the sequence to be analyzed on the single-stranded nucleic acid target except at the interrogation position, located at the 5'-end of the probes in a position adjacent to the attachment site of the label, where each of the four nucleic bases were incorporated. This work allowed the selection of probes showing, upon their hybridization with the target sequence, good discrimination between the matched and the mismatched duplexes under non-stringent conditions, with the mismatched duplexes being more fluorescent than the perfectly matched ones.