New cyanine-oligonucleotide conjugates: relationships between chemical structures and properties

Because the influence of the chemical structure of monomethine cyanine-oligo-2'-deoxyribonucleotide (ODN) conjugates on their binding and fluorescence properties has remained largely undetermined, we synthesized and studied a wide range of conjugates with various structural patterns. Different cyanine dyes such as thiocyanine, quinocyanine, and thiazole orange isomers were obtained. In the case of unsymmetrical cyanines, the linker was attached to either the quinoline or the benzothiazole nucleus. The influence of the ODN counterpart was evaluated by linking the cyanines to the 5'-end or to an internucleotidic phosphate. In the first case, the influence of neighboring nucleic bases was studied, whereas in the second, the stereochemical configuration at the phosphorus atom bearing the cyanine was investigated. We report here on relationships between the structures of the dyes and conjugates and some of their properties, such as the stability and fluorescence changes observed on their hybridization with the target sequence. This study provides useful information towards the design of ODN-cyanine conjugates.     

Sequence-specific fluorescence detection of DNA by polyamide-thiazole orange conjugates

Fluorescent methods to detect specific double-stranded DNA sequences without the need for denaturation may be useful in the field of genetics. Three hairpin pyrrole-imidazole polyamides 2-4 that target their respective sequences 5'-WGGGWW-3', 5'-WGGCCW-3', and 5'-WGWWCW-3' (W = A or T) were conjugated to thiazole orange dye at the C-termini to examine their fluorescence properties in the presence and absence of match duplex DNA. The conjugates fluoresce weakly in the absence of DNA but showed significant enhancement (>1000-fold) upon the addition of 1 equiv of match DNA and only slight enhancement with the addition of mismatch DNA. The polyamide-dye conjugates bound specific DNA sequences with high affinity (Ka > 10(8) M(-1)) and unwound the DNA duplex through intercalation (unwinding angle, phi, approximately 8 degrees). This new class of polyamides provides a method to specifically detect DNA sequences without denaturation.     

噻唑橙类菁染料与生物大分子结合的光谱特性的研究

研究了两个噻唑橙类菁染料TO-1和TO-2与核酸、蛋白质结合的光谱特性.发现染料喹啉环中氮上取代基对染料标记生物大分子的荧光特性有着很大的影响.羧基的引入,使染料TO-2与核酸结合后的荧光性能下降,但同时也提高了它与蛋白质结合的光物理性能.研究表明:在喹啉环氮上引入适当的亲水性取代基,可以使噻唑橙类菁染料的应用范围拓展到蛋白质标记领域.并初步研究了蛋白质浓度的变化对染料TO-2的荧光光谱的影响以及TO-2在不同pH值的蛋白质溶液中的荧光性质.     

Thermodynamic versus kinetic products of DNA alkylation as modeled by reaction of deoxyadenosine.

Alkylating agents that react through highly electrophilic quinone methide intermediates often express a specificity for the weakly nucleophilic exocyclic amines of deoxyguanosine (dG N(2)) and deoxyadenosine (dA N(6)) in DNA. Investigations now indicate that the most nucleophilic site of dA (N1) preferentially, but reversibly, conjugates to a model ortho-quinone methide. Ultimately, the thermodynamically stable dA N(6) isomer accumulates by trapping the quinone methide that is transiently regenerated from collapse of the dA N1 adduct. Alternative conversions of the dA N1 to the dA N(6) derivative by a Dimroth rearrangement or other intramolecular processes are not competitive under neutral conditions, as demonstrated by studies with [6-(15)N]-dA. Both a model quinone methide precursor and its dA N1 adduct yield a similar profile of deoxynucleoside products when treated with an equimolar mixture of dC, dA, dG, and T. Consequently, the most readily observed products of DNA modification resulting from reversible reactions may reflect thermodynamic rather than kinetic selectivity.     

Hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine: kinetics and mechanisms for the cleavage, desulfurization, and isomerization of the internucleosidic linkage

The hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine [3',5'-Up(s)2U] were followed by HPLC over a wide pH range at 363.2 K. Under acidic and neutral conditions, three reactions compete: (i) desulfurization to a mixture of the (Rp)- and (Sp)-diastereomers of the corresponding 3',5'- and 2',5'-phosphoromonothioates [3',5'- and 2',5'-Up(s)U], which are subsequently desulfurized to a mixture of uridylyl(3',5')- and -(2',5')uridine [3',5'- and 2',5'-UpU], (ii) isomerization to 2',5'-Up(s)2U, and (iii) cleavage to uridine, in all likelihood via a 2',3'-cyclic phosphorodithioate (2',3'-cUMPS2). Under alkaline conditions (pH > 8), only a hydroxide ion catalyzed hydrolysis to uridine via 2',3'-cUMPS2 takes place. At pH 3-7, all three reactions are pH-independent, the desulfurization being approximately 1 order of magnitude faster than the cleavage and isomerization. At pH < 3, all the reactions are hydronium ion catalyzed. On going to very acidic solutions, the cleavage gradually takes over the desulfurization and isomerization. Accordingly, the cleavage overwhelmingly predominates at pH < 0. The overall hydrolytic stability of 3',5'-Up(s)2U is comparable to that of (Sp)- and (Rp)-3',5'-Up(s)U (and to that of 3',5'-UpU, except at pH < 2). The rate of the hydroxide ion catalyzed hydrolysis of 3',5'-Up(s)2U is 37% and 53% of that of (Sp)- and (Rp)-3',5'-Up(s)U, respectively. The reactions, however, differ with the respect of the product accumulation. While the phosphoromonothioates produce a mixture of 2'- and 3'-thiophosphates as stable products, 3',5'-Up(s)2U is hydrolyzed to uridine without accumulation of the corresponding dithiophosphates. At pH < 3, where the hydrolysis is hydronium ion catalyzed, the kinetic thio-effect of the second thio substitution is small: under very acidic conditions (Ho -0.69), (Sp)-3',5'-Up(s)U reacts 1.6 times as fast as 3',5'-Up(s)2U, but the reactivity difference decreases on going to less acidic solutions. In summary, the hydrolytic stability of 3',5'-Up(s)2U closely resembles that of the corresponding phosphoromonothioate. While replacing one of the nonbridging phosphate oxygens of 3',5'-UpU with sulfur stabilizes the phosphodiester bond under acidic conditions by more than 1 order of magnitude, the replacement of the remaining nonbridging oxygen has only a minor influence on the overall hydrolytic stability.     

Solvent and linker influences on AQ(*-)/dA(*+) charge-transfer state energetics and dynamics in anthraquinonyl-linker-deoxyadenosine conjugates

The goal of this work is to produce high yields of long-lived AQ(*-)/dA(*+) charge transfer (CT) excited states (or photoproducts). This goal fits within a larger context of trying generally to produce high yields of long-lived CT excited states within DNA nucleoside conjugates that can be incorporated into DNA duplexes. Depending upon the energetics of the anthraquinonyl (AQ) (3)(pi,pi) state as well as the reduction potentials of the subunits in particular anthraquinonyl-adenine conjugates, CT quenching of the AQ (3)(pi,pi*) state may or may not occur in polar organic solvents. In MeOH, bis(3',5'-O-acetyl)-N(6)-(anthraquinone-2-carbonyl)-2'-deoxyadenosine (AQCOdA) behaves as intended and forms a (3)(AQ(*-)/dA(*+)) CT state with a lifetime of 3 ns. However, in nonpolar THF the AQ(*-)/dA(*+) CT states of AQCOdA are too high in energy to be formed, and in DMSO a (1)(AQ(*-)/dA(*+)) CT state is formed but lives only 6 ps. Although the lowest energy excited state for AQCOdA in MeOH is a (3)(AQ(*-)/dA(*+)) CT state, for N(6)-(anthraquinone-2-methylenyl)-2'-deoxyadenosine (AQMedA) in the same solvent it is a (3)(pi,pi*) state. Changing the linking carbonyl in AQCOdA to methylene in AQMedA makes the anthraquinonyl subunit harder to reduce by 166 mV. This raises the energy of the (3)(AQ(*-)/dA(*+)) CT state above that of the (3)(pi,pi*) in AQMedA. The conclusion is that anthraquinonyl-dA conjugates will not have lowest energy AQ(*-)/dA(*+) CT states in polar organic solvents unless the anthraquinonyl subunit is also substituted with an electron-withdrawing group that raises the AQ-subunit's reduction potential above that of AQ. A key finding in this work is that the lifetime of the (3)(AQ(*-)/dA(*+)) CT excited state (ca. 3 ns) is ca. 500-times longer than that of the corresponding (1)(AQ(*-)/dA(*+)) CT excited state (ca. 6 ps).'     

Enantioselective self-assembly of bimetallic [Mn(II) (delta)-Cr(III)(C2O4)3]- and [Mn(II) (lambda)-Cr(III)(C2O4)3]- layered anionic networks templated by the optically active (Rp)- and (Sp)-[1-CH2N(n-C3H7)3-2-CH3-C5H3Fe-C5H5]+ ions

The ferrocenic ammonium (Rp)- and (Sp)-[1-CH2NR(3-)-2-CH3-C5H3Fe-C5H5] iodide salts with R=CH3, C2H5, n-C3H7, n-C4H9, were synthesized starting from the (Rp)- and (Sp)-[1-CH2N(CH3)2-2-CH3-C5H3Fe-C5H5] amines obtained in their optically active forms through asymmetric cyclopalladation of [C5H5Fe-C5H4CH2N(CH3)2]. 1H NMR studies of these planar chiral 1,2-disubstituted ferrocenic ammonium iodide salts in the presence of the (Delta)-(tris(tetrachlorobenzenediolato)phosphate(V) anion), [(Delta)-Trisphat] support the formation of specific diastereomeric ion pairs. Such intermolecular interactions can be related to the self-assembly of the two-dimensional optically active compounds [[(Sp)-1-CH2N(n-C3H7)3-2-CH3-C5H3Fe-C5H5][Mn (Delta)-Cr(C2O4)3]] and [[(Rp)-1-CH2N(n-C3H7)3-2-CH3-C5H3Fe-C5H5][Mn (Lambda)-Cr(C2O4)3]] starting from the resolved (Rp)- and (Sp)-[1-CH2N(n-C3H7)3-2-CH3-C5H3Fe-C5H5]+ ion associated to the racemic anionic building block rac-[Cr(C2O4)3]3- and Mn2+. Both enantiomeric forms of the networks behave as ferromagnets with a Curie temperature of 5.7 K.     

Thiazole orange-peptide conjugates: sensitivity of DNA binding to chemical structure

[structure: see text] Derivatives of the highly fluorescent and DNA-binding dye thiazole orange (TO) are described that feature appended peptides. Functionalization of TO can be achieved at either of the endocyclic nitrogens, and the photophysical properties and DNA-binding modes are sensitive to the position of the tethered peptide. A series of TO-peptide conjugates are described, demonstrating the utility of a solid-phase synthesis approach to their preparation and illustrating how the photophysical and DNA-binding properties of the compounds are influenced by chemical structure.     

Highly fluorescent supramolecular gels with chirality transcription through hydrogen bonding

A highly fluorescent organogel with transparency was formed through a hydrogen (H)-bonding interaction between a non-fluorescent and achiral 2-(3',5'-bis-trifluoromethyl-biphenyl-4-yl)-3-(4-pyridin-4-yl-phenyl)-acrylonitrile (CN-TFMBPPE) monomer and chiral sergeant l-tartaric acid (TA) (or d-TA), with gel formation being accompanied by a drastic fluorescence enhancement as well as chirality induction.     

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     

基于荧光探针噻唑橙与核酸适体构象变化的钾离子检测

建立了一种基于核酸适体(Aptamer)构象效应和荧光探针噻唑橙(TO)为荧光分子开关进行钾离子检测的光学方法.室温下钾离子可与Aptamer结合形成G-四面体结构,使双链解链变为四面体结构和单链,从而导致TO荧光强度降低.考察了TO浓度、反应温度及反应时间的影响.在最佳实验条件下,钾离子浓度在1.0×10-6 ～2....     

7-Deazapurine and 8-aza-7-deazapurine nucleoside and oligonucleotide pyrene "click" conjugates: synthesis, nucleobase controlled fluorescence quenching, and duplex stability

7-Deazapurine and 8-aza-7-deazapurine nucleosides related to dA and dG bearing 7-octadiynyl or 7-tripropargylamine side chains as well as corresponding oligonucleotides were synthesized. "Click" conjugation with 1-azidomethyl pyrene (10) resulted in fluorescent derivatives. Octadiynyl conjugates show only monomer fluorescence, while the proximal alignment of pyrene residues in the tripropargylamine derivatives causes excimer emission. 8-Aza-7-deazapurine pyrene "click" conjugates exhibit fluorescence emission much higher than that of 7-deazapurine derivatives. They are quenched by intramolecular charge transfer between the nucleobase and the dye. Oligonucleotide single strands decorated with two "double clicked" pyrenes show weak or no excimer fluorescence. However, when duplexes carry proximal pyrenes in complementary strands, strong excimer fluorescence is observed. A single replacement of a canonical nucleoside by a pyrene conjugate stabilizes the duplex substantially, most likely by stacking interactions: 6-12 °C for duplexes with a modified "adenine" base and 2-6 °C for a modified "guanine" base. The favorable photophysical properties of 8-aza-7-deazapurine pyrene conjugates improve the utility of pyrene fluorescence reporters in oligonucleotide sensing as these nucleoside conjugates are not affected by nucleobase induced quenching.     

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.     

Oligonucleotide-conjugated thiazole orange probes as "light-up" probes for messenger ribonucleic acid molecules in living cells.

"Light-up" probes, icosa-alpha-thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous solutions, the icosa-alpha-thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes into adherent fibroblasts resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.     

Near-Infrared Hydrophobic Probes as Molecular Light Switch for CMC Determination of Triton X-100 Solution

The fluorescence behavior of two near-infrared (NIR) chromophores with linear alkyl chains of different lengths, 2-[4′chloro-7′(3″ethyl-2″benzothiaolinylidene)-3′,5′-(1″′3′″-propanediyl)-1′3′,5′-heptantriene-1′-yL]-3-ethylbenzothiazolium iodide (Probe Ⅰ) and 2-[4′chloro-7′(3″hexadecyl-2″benzothiazolinylidene)-3′,5′-(1′″,3′″-propanediyl)-1′,3′,5′-heptantriene-1′-yl]-3-ethylbenzothiazolium iodide (Probe Ⅱ), in aqueous solution containing different concentrations of surfactants was studied. The fluorescence of the probe with a short chain (probe Ⅰ) was completely quenched in water and aqueous solution containing a low concentration (below the critical micelle concentration,CMC) of surfactant Triton X-100. However, the fluorescence reappeared and reached maximum rapidly once the concentration of the surfactant approached the CMC. The probe with a long chain (probe II) displayed a similar fluorescence behavior but more dramatically fluorescent recovery in Triton X-100 system, which gave a direct in- dication for the micelle forming process and provided a simple method for the determination of the critical micelle concentration of the surfactant. The CMC values determined by this method were in good agreement with those obtained by other techniques. The fluorescence behavior of the two probes in other surfactant systems was also investigated.     

FRET templated by G-quadruplex DNA: a specific ternary interaction using an original pair of donor/acceptor partners

G-quadruplex represents a suitable scaffold for FRET (fluorescence resonance energy transfer) since its two external quartets offer two well-defined binding sites for concomitant trapping of donor/acceptor partners. Combining selective G-quadruplex binders (macrocyclic bis(quinacridine) BOQ(1) or monomeric quinacridine MMQ(1), donor) with a highly fluorescent DNA probe (thiazole orange, acceptor), we designed a structure-specific FRET-system based on an unprecedented noncovalent ternary complex. This system could be potentially usable as a signature for quadruplex-DNA conformation in solution, but also might offer a unique means for observing cation and ligand binding influence on quadruplex topology.     

The pK(a) of the internucleotidic 2'-hydroxyl group in diribonucleoside (3'-->5') monophosphates

Ionization of the internucleotidic 2'-hydroxyl group in RNA facilitates transesterification reactions in Group I and II introns (splicing), hammerhead and hairpin ribozymes, self-cleavage in lariat-RNA, and leadzymes and tRNA processing by RNase P RNA, as well as in some RNA cleavage reactions promoted by ribonucleases. Earlier, the pK(a) of 2'-OH in mono- and diribonucleoside (3'-->5') monophosphates had been measured under various nonuniform conditions, which make their comparison difficult. This work overcomes this limitation by measuring the pK(a) values for internucleotidic 2'-OH of eight different diribonucleoside (3'-->5') monophosphates under a set of uniform noninvasive conditions by 1H NMR. Thus the pK(a) is 12.31 (+/-0.02) for ApG and 12.41 (+/-0.04) for ApA, 12.73 (+/-0.04) for GpG and 12.71 (+/-0.08) for GpA, 12.77 (+/-0.03) for CpG and 12.88 (+/-0.02) for CpA, and 12.76 (+/-0.03) for UpG and 12.70 (+/-0.03) for UpA. By comparing the pK(a)s of the respective 2'-OH of monomeric nucleoside 3'-ethyl phosphates with that of internucleotidic 2'-OH in corresponding diribonucleoside (3'-->5') monophosphates, it has been confirmed that the aglycons have no significant effect on the pK(a) values of their 2'-OH under our measurement condition, except for the internucleotidic 2'-OH of 9-adeninyl nucleotide at the 5'-end (ApA and ApG), which is more acidic by 0.3-0.4 pK(a) units.     

A general RNA-capping ribozyme retains stereochemistry during cap exchange

Numerous natural and artificial ribozymes have been shown to facilitate reactions that invert stereochemistry. Here, we demonstrate that an RNA-capping ribozyme retains stereochemistry at a phosphorus reaction center. The ribozyme synthesizes a broad range of 5'-5' RNA caps by exchanging phosphate groups around the alpha-phosphate found at the 5' terminus of the ribozyme. A ribozyme prepared with an Rp adenosine(5')alpha-thiotetraphosphate cap was found to exchange this cap for an Rp 4-thiouridine(5')alpha-thiotetraphosphate cap when incubated with 4-thiouridine triphosphate. The same Rp capped construct, when incubated with [gamma-(32)P]-ATP, exchanged the unlabeled ATP for a radiolabeled one while maintaining the same stereoconfiguration. In contrast, ribozymes prepared with an Sp cap failed to react even in the presence of thiophilic metal ions such as manganese. The kinetics of capping was also unusual as compared to inverting ribozymes. When the ribozyme was prepared with a triphosphate, capping was found to follow Michaelis-Menten-type kinetics even though the rate of pyrophosphate release was completely independent of nucleotide substrate concentration. Interestingly, the rate of capping and hydrolysis, when summed, was found to be indistinguishable from the rate of pyrophosphate release, indicating that an early rate-limiting step precedes both capping and hydrolysis. Together the retention of stereochemistry and kinetics imply that capping utilizes two inverting chemical steps that are separated by the transient formation of a rate-limiting covalent intermediate. As all protein enzymes that mediate similar capping reactions utilize a covalent intermediate, chemical necessity may have strongly guided the evolution of both protein and RNA-capping catalysts.     

A simple, high-resolution method for establishing DNA binding affinity and sequence selectivity.

Full details of the development of a simple, nondestructive, and high-throughput method for establishing DNA binding affinity and sequence selectivity are described. The method is based on the loss of fluorescence derived from the displacement of ethidium bromide or thiazole orange from the DNA of interest or, in selected instances, the change in intrinsic fluorescence of a DNA binding agent itself and is applicable for assessing relative or absolute DNA binding affinities. Enlisting a library of hairpin deoxyoligonucleotides containing all five base pair (512 hairpins) or four base pair (136 hairpins) sequences displayed in a 96-well format, a compound's rank order binding to all possible sequences is generated, resulting in a high-resolution definition of its sequence selectivity using this fluorescent intercalator displacement (FID) assay. As such, the technique complements the use of footprinting or affinity cleavage for the establishment of DNA binding selectivity and provides the information at a higher resolution. The merged bar graphs generated by this rank order binding provide a qualitative way to compare, or profile, DNA binding affinity and selectivity. The 96-well format assay (512 hairpins) can be conducted at a minimal cost (presently ca. $100 for hairpin deoxyoligonucleotides/assay with ethiduim bromide or less with thiazole orange), with a rapid readout using a fluorescent plate reader (15 min), and is adaptable to automation (Tecan Genesis Workstation 100 robotic system). Its use in generating a profile of DNA binding selectivity for several agents including distamycin A, netropsin, DAPI, Hoechst 33258, and berenil is described. Techniques for establishing binding constants from quantitative titrations are compared, and recommendations are made for use of a Scatchard or curve fitting analysis of the titration binding curves as a reliable means to quantitate the binding affinity.     

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).