Discrimination of single-nucleotide alterations by G-specific fluorescence quenching

A new strategy for the detection of single-base alterations through fluorescence quenching by guanine (G) is described. We have devised a novel base-discriminating fluorescent (BDF) nucleoside, 4'PyT, that contains a pyrenecarboxamide fluorophore at the thymidine sugar's C4'-position. 4'PyT-containing oligodeoxynucleotides only exhibited enhanced fluorescence in response to the presence of a complementary adenine base. In contrast, the fluorescence of mismatched duplexes containing 4'PyT/N base pairs (N = C, G, or T) was considerably weaker. This highly A-selective fluorescence was a product of guanine-specific quenching efficiency; when the complementary base to 4'PyT was a mismatch, the pyrenecarboxamide fluorophore was able to interact intimately with neighboring G bases (the most likely interaction in the case of intercalation), so effective quenching by the G bases occurred in the mismatched duplexes. In contrast, duplexes containing 4'PyT/A base pairs exhibited strong emission, since in this case the fluorophores were positioned in the minor groove and able to escape fluorescence quenching by the G bases.     

Binding of helix-threading peptides to E. coli 16S ribosomal RNA and inhibition of the S15-16S complex

Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.     

Guanidinoneomycin B recognition of an HIV-1 RNA helix

Aminoglycoside antibiotics are small-molecule drugs that bind RNA. The affinity and specificity of aminoglycoside binding to RNA can be increased through chemical modification, such as guanidinylation. Here, we report the binding of guanidinoneomycin B (GNB) to an RNA helix from the HIV-1 frameshift site. The binding of GNB increases the melting temperature (T(m)) of the frameshift-site RNA by at least 10 degrees C, to a point at which a melting transition is not even observed in 2 M urea. A structure of the complex was obtained by using multidimensional heteronuclear NMR spectroscopic methods. We also used a novel paramagnetic-probe assay to identify the site of GNB binding to the surface of the RNA. GNB makes major-groove contacts to two sets of Watson-Crick bases and is in van der Waals contact with a highly structured ACAA tetraloop. Rings I and II of GNB fit into the major groove and form the binding interface with the RNA, whereas rings III and IV are exposed to the solvent and disordered. The binding of GNB causes a broadening of the major groove across the binding site.     

Thermodynamic analysis of nylon nucleic acids

The stability and structure of nylon nucleic acid duplexes with complementary DNA and RNA strands was examined. Thermal denaturing studies of a series of oligonucleotides that contained nylon nucleic acids (1-5 amide linkages) revealed that the amide linkage significantly enhanced the binding affinity of nylon nucleic acids towards both complementary DNA (up to 26 degrees C increase in the thermal transition temperature (T(m)) for five linkages) and RNA (around 15 degrees C increase in T(m) for five linkages) compared with nonamide linked precursor strands. For both DNA and RNA complements, increasing derivatization decreased the melting temperatures of uncoupled molecules relative to unmodified strands; by contrast, increasing lengths of coupled copolymer raised T(m) from less to slightly greater than T(m) of unmodified strands. Thermodynamic data extracted from melting curves and CD spectra of nylon nucleic acid duplexes were consistent with loss of stability due to incorporation of pendent groups on the 2'-position of ribose and recovery of stability upon linkage of the side chains.     

New approaches toward recognition of nucleic acid triple helices

A DNA duplex can be recognized sequence-specifically in the major groove by an oligodeoxynucleotide (ODN). The resulting structure is a DNA triple helix, or triplex. The scientific community has invested significant research capital in the study of DNA triplexes because of their robust potential for providing new applications, including molecular biology tools and therapeutic agents. The triplex structures have inherent instabilities, however, and the recognition of DNA triplexes by small molecules has been attempted as a means of strengthening the three-stranded complex. Over the decades, the majority of work in the field has focused on heterocycles that intercalate between the triplex bases. In this Account, we present an alternate approach to recognition and stabilization of DNA triplexes. We show that groove recognition of nucleic acid triple helices can be achieved with aminosugars. Among these aminosugars, neomycin is the most effective aminoglycoside (groove binder) for stabilizing a DNA triple helix. It stabilizes both the TAT triplex and mixed-base DNA triplexes better than known DNA minor groove binders (which usually destabilize the triplex) and polyamines. Neomycin selectively stabilizes the triplex (TAT and mixed base) without any effect on the DNA duplex. The selectivity of neomycin likely originates from its potential and shape complementarity to the triplex Watson-Hoogsteen groove, making it the first molecule that selectively recognizes a triplex groove over a duplex groove. The groove recognition of aminoglycosides is not limited to DNA triplexes, but also extends to RNA and hybrid triple helical structures. Intercalator-neomycin conjugates are shown to simultaneously probe the base stacking and groove surface in the DNA triplex. Calorimetric and spectrosocopic studies allow the quantification of the effect of surface area of the intercalating moiety on binding to the triplex. These studies outline a novel approach to the recognition of DNA triplexes that incorporates the use of noncompeting binding sites. These principles of dual recognition should be applicable to the design of ligands that can bind any given nucleic acid target with nanomolar affinities and with high selectivity.     

Probing the Structural Properties of DNA/RNA Grooves with Sterically Restricted Phosphonium Dyes: Screening of Dye Cytotoxicity and Uptake

To explore in greater detail the recently reported rare kinetic differentiation between homo‐polymeric and alternating AT‐DNA sequences by using sterically restricted phosphonium dyes that form dimers within the DNA minor groove, new analogues were prepared in which the quinolone phosphonium moiety was kept constant, while the size and hydrogen bonding properties of the rest of the molecule were varied. Structure–activity relationship studies revealed that a slight increase in length by an additional methylene unit results in loss of kinetic AT selectivity, but yielded an AT‐selective fluorescence response. These DNA/RNA‐groove‐bound dyes combine very low cytotoxicity with efficient cellular uptake and intriguingly specific fluorescent marking of mitochondria. In contrast to longer analogues, a decrease in length (by methylene unit removal) and rearrangement of positive charge resulted in dyes that had switched to the intercalative binding mode to GC DNA/dsRNA but that still form dimers in the minor groove of AT sequences, consequently yielding a significantly different chiro‐optical response. The latter dyes also revealed strongly selective antiproliferative activity toward HeLa cancer cells.     

An Inverse Substrate Orientation for the Regioselective Acylation of 3′,5′‐Diaminonucleosides Catalyzed by Candida antarctica lipase B?

Candida antarctica lipase B (CAL-B) catalyzes the regioselective acylation of natural thymidine with oxime esters and also the regioselective acylation of an analogue, 3',5'-diamino-3',5'-dideoxythymidine with nonactivated esters. In both cases, acylation favors the less hindered 5'-position over the 3'-position by upto 80-fold. Computer modeling of phosphonate transition-state analogues for the acylation of thymidine suggests that CAL-B favors acylation of the 5'-position because this orientation allows the thymine ring to bind in a hydrophobic pocket and forms stronger key hydrogen bonds than acylation of the 3'-position. On the other hand, computer modeling of phosphonamidate analogues of the transition states for acylation of either the 3'- or 5'-amino groups in 3',5'-diamino-3',5'-dideoxythymidine shows similar orientations and hydrogen bonds and, thus, does not explain the high regioselectivity. However, computer modeling of inverse structures, in which the acyl chain binds in the nucleophile pocket and vice versa, does rationalize the observed regioselectivity. The inverse structures fit the 5'-, but not the 3'-intermediate thymine ring, into the hydrophobic pocket, and form a weak new hydrogen bond between the O-2 carbonyl atom of the thymine and the nucleophile amine only for the 5'-intermediate. A water molecule might transfer a proton from the ammonium group to the active-site histidine. As a test of this inverse orientation, we compared the acylation of thymidine and 3',5'-diamino-3',5'-dideoxythymidine with butyryl acyl donors and with isosteric methoxyacetyl acyl donors. Both acyl donors reacted at equal rates with thymidine, but the methoxyacetyl acyl donor reacted four times faster than the butyryl acyl donor with 3',5'-diamino-3',5'-dideoxythymidine. This faster rate is consistent with an inverse orientation for 3',5'-diamino-3',5'-dideoxythymidine, in which the ether oxygen atom of the methoxyacetyl group can form a similar hydrogen bond to the nucleophilic amine. This combination of modeling and experiments suggests that such lipase-catalyzed reactions of apparently close substrate analogues like alcohols and amines might follow different pathways.     

Pyrenemethyl ara-uridine-2'-carbamate: a strong interstrand excimer in the major groove of a DNA duplex

The synthesis of new nucleoside derivatives, ara-uridine-2'-carbamates, and their incorporation into synthetic DNA oligomers is described. The modification directs ligands into the major groove of duplex DNA and somewhat destabilizes the duplexes of modified oligonucleotides with complementary DNA or RNA. In the case of pyrenemethyl carbamate modification in DNA-DNA duplexes, the destabilization is considerably reduced. The pyrenemethyl derivative also shows remarkable spectral properties: a "reversed" absorbance change for pyrene at 350 nm in the course of denaturation of the DNA duplex, as compared to the change seen in the nucleotide absorbance at 260 nm. This derivatization also causes pronounced sequence-dependent excimer formation in the major groove.     

Effect of north bicyclo[3.1.0]hexane 2'-deoxy-pseudosugars on RNA interference: a novel class of siRNA modification

North bicyclo methanocarba thymidine (T(N)) nucleosides were substituted into siRNAs to investigate the effect of bicyclo[3.1.0]hexane 2'-deoxy-pseudosugars on RNA interference activity. Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery. We studied the effect of the T(N) modification in a screen involving residue-specific changes in an siRNA targeting Renilla luciferase and we applied the most effective pattern of modification to the knockdown of murine tumor necrosis factor (TNF-α). We also showed that incorporation of T(N) units into siRNA duplexes increased their thermal stabilities, substantially enhanced serum stabilities, and decreased innate immunostimulation. Comparative RNAi studies involving the T(N) substitution and locked nucleic acids (LNAs) showed that the gene-silencing activities of T(N) -modified siRNAs were comparable to those obtained with the LNA modification. An advantage of the North 2'-deoxy-methanocarba modification is that it may be explored further in the future by changing the 2'-position. The results from these studies suggest that this modification might be valuable for the development of siRNAs for therapeutic applications.     

B-ring-modified isocombretastatin A-4 analogues endowed with interesting anticancer activities

A novel class of isocombretastatin A-4 (isoCA-4) analogues with modifications at the 3'-position of the B-ring by replacement with C-linked substituents was studied. Exploration of the structure-activity relationships of theses analogues led to the identification of several compounds that exhibit excellent antiproliferative activities in the nanomolar concentration range against H1299, MDA-MB231, HCT116, and K562 cancer cell lines; they also inhibit tubulin polymerization with potency similar to that of isoCA-4. 1,1-Diarylethylenes 8 and 17, respectively with (E)-propen-3-ol and propyn-3-ol substituents at the 3'-position of the B-ring, proved to be the most active in this series. Both compounds led to the arrest of various cancer cell lines at the G(2) /M phase of the cell cycle and strongly induced apoptosis. Docking of compounds 8 and 17 in the colchicine binding site indicated that their C3' substituents guide the positioning of the B-ring in a manner different from that observed for isoCA-4.     

2,2′,7,7′-Tetrahydroxy-1,1′-Binaphthyl: A Versatile Chiral Spacer for Monotopic and Ditopic Cyclophane Hosts with Apolar Binding Sites

Derivatives of 2,2′,7,7′-tetrahydroxy-1,1′-binaphthyl ( 5 ) are synthetically readily available chiral shapes with a major and a minor groove. Previous work by Cram et al. has shown that efficient chiral crown ether binding sites can be constructed at the binaphthyl minor groove. The major groove is almost twice as wide and possesses suitable dimensions to shape flat apolar binding sites in chiral cyclophane hosts. The eight-step syntheses of the two novel cyclophanes 6 and 7 are described. The apolar cavities of these hosts are formed by bridging with two linear four-carbon chains the 7,7′-oxygens at the major groove of 5 to the two phenolic oxygens of a 4,4-bis(4-hydroxy-3,5-dimethylphenyl)piperidine unit. ¹H NMR complexation studies show that 1:1 complexes of 6 and 7 with 6-methoxy-2-naphthonitrile and other naphthalene derivatives in aqueous methanol are as stable as the previously reported complexes of bis(diphenylmethane)-hosts. The different solvation characteristics of cation and apolar binding are demonstrated with the ditopic host 7 . In addition to the apolar binding site at the major groove, 7 also possesses a crown ether binding site at the minor groove of the binaphthyl spacer. By changing the methanol content of a water-methanol mixture, this host can be switched from an efficient binder of neutral naphthalene guests to a good host for potassium cations. The activities of the two binding sites in 7 are almost entirely independent of each other; consequently, the binding of a naphthalene guest in the 1:1:1 (host-naphthalene-K⁺) complex is nearly as strong as in the 1:1 complex. The methyl ester of (S)-naproxen forms stable complexes with the racemic hosts 6 and 7 . The ¹H NMR spectra of these complexes show considerably different shifts for the aromatic resonances of the host enantiomers. These differences indicate the formation of diastereomeric complexes of different geometries or stabilities.     

Remote interactions explain the unusual regioselectivity of lipase from Pseudomonas cepacia toward the secondary hydroxyl of 2'-deoxynucleosides

Lipase from Pseudomonas cepacia (PCL) surprisingly favors acylation of the secondary hydroxyl at the 3'-position over the primary hydroxyl at the 5'-position in 2'-deoxynucleosides by up to >98:1. Catalytically productive tetrahedral intermediate analogues for both orientations were found by molecular modeling. However, acylation of the 3'-hydroxyl places the thymine base in the alternate hydrophobic pocket of PCL's substrate-binding site where it can hydrogen bond to the side-chain hydroxyls of Tyr23 and Tyr29 and the main chain carbonyl of Leu17. Conversely, acylation of the 5'-hydroxyl leaves the thymine base in the solvent where there is no favorable binding to the enzyme. We propose that these remote stabilizing interactions between the thymine base and PCL's substrate-binding site stabilize the 3'-acylation transition state and thus account for the unusual regioselectivity.     

香豆素类染料的荧光光谱性能及应用性能研究

研究了9只香豆素类荧光染料(Ⅰ-Ⅶ)的光谱性能和应用性能。香豆素类荧光染料在5’-位有取代基可使最大可见吸收波长和荧光发射波长产生红移;当共轭双键长度增加,可使染料颜色变深(V:544,VI:552)。这9只染料用于涤纶染色有很好的应用性能,差别不大;嗯唑环结构(Ⅰ、Ⅱ、Ⅲ)和喹唑酮结构(Ⅵ)的染料耐光牢度明显好于噻唑环结构(Ⅴ)和咪唑环结构(Ⅶ)的染料。     

基于遗传算法的干气密封双向槽统一模型与参数优化

通过提出表征能力更强的新型结构模型和引入全局寻优能力更强的智能优化算法以提高双向旋转槽干气密封的稳态密封性能。在综合分析国内外干气密封典型双向旋转槽结构特点的基础上，提出一种动压槽倾角可变的双向旋转槽统一模型。建立双向旋转槽干气密封的几何模型和数学模型，采用有限差分法求解端面膜压控制方程，获得开启力和气膜刚度等稳态性能参数。分析了动压槽倾斜角对干气密封稳态性能的影响规律，对比分析了不同速度条件下单因素优化、迭代优化和遗传算法优化等三种优化方法对提高双向旋转槽干气密封稳态性能的作用。结果表明：相较于以往双向树形槽干气密封单因素优化的结果，基于遗传算法的统一模型槽干气密封获得的开启力和气膜刚度显著提高，最大增幅分别达到6%和55%；在高速条件下，上游动压槽螺旋角为锐角，下游动压槽螺旋角为钝角的似机翼形双向旋转槽干气密封具有最大的开启力和气膜刚度。     

Synthesis and evaluation of acyl-chain- and galactose-6'-modified analogues of α-GalCer for NKT cell activation

α-GalCer is an immunostimulating glycolipid that binds to CD1d molecules and activates invariant natural killer T (iNKT) cells. Here we report a scaled-up synthesis of α-GalCer analogues with modifications in the acyl side chain and/or at the galactose 6'-position, together with their evaluation in vitro and in vivo. Analogues containing 11-phenylundecanoyl acyl side chains with aromatic substitutions (14, 16-21) and Gal-6'-phenylacetamide-substituted α-GalCer analogues bearing p-nitro- (32), p-tert-butyl (34), or o-, m-, or p-methyl groups (40-42) displayed higher IFN-γ/IL-4 secretion ratios than α-GalCer in vitro. In mice, compound 16, with an 11-(3,4-difluorophenyl)undecanoyl acyl chain, induced significant proliferation of NK and DC cells, which should be beneficial in killing tumors and priming the immune response. These new glycolipids might prove useful as adjuvants or anticancer agents.     

一种自适应柱状密封气膜特性分析

提出一种自适应柱状气膜密封,构建气膜-密封环为研究对象,考虑了偏心间隙发散问题,并定义Rayleigh台阶对气膜周期的突变性,建立了气膜雷诺方程和膜厚变化函数关系式的动力润滑数值模型,获得了摩擦副气膜润滑特性,研究结果表明：气膜力随转速和压力升高而增加,泄漏量随压力升高而增加但是随转速增加而下降,说明密封内部压力流占主导地位。随后,利用曲面槽型雕刻技术,结合高速试验台对密封进行测试,结果表明：理论计算模型与试验结果吻合度较高,斜槽区域存在更多擦痕与磨损,说明斜槽浮动性弱于直列槽;但斜槽的切向流动小于直列槽,导致斜槽泄漏量小于直列槽。该研究成果为自适应柱状气膜密封的气体流动规律和应用提供了理论基础。     

干气密封螺旋槽几何参数优选交互影响

为解决干气密封（DGS）端面型槽优化中未考虑型槽几何参数之间对目标函数交互影响而导致几何参数优化结果不准确的问题,研究了不同速度条件下DGS螺旋槽中其他几何参数对某一几何参数优选值的交互影响。基于气体润滑理论,建立了螺旋槽DGS的几何模型和数学模型,数值求解获得端面膜压分布和稳态性能参数,定义了综合表征螺旋槽几何结构的特征参数。以气膜刚度最大为优选目标,获得了不同速度条件下DGS螺旋槽中某一几何参数优选值随其他几何参数的变化规律。结果表明：DGS螺旋槽各几何参数两两之间呈现出复杂的交互影响关系,不过总体符合趋向于降低槽内实际通流面积、槽长宽比和槽长深比3个综合参数变化幅值的原则;与单因素优化方法相比,在给定算例参数条件下所提出的考虑几何参数交互影响的优化方法所获得气膜刚度最优值提高11.4%。     

收缩型芯内抽芯和旋转型芯脱螺纹注塑模

根据放大镜框的结构特点,设计了镜框注塑模。论述了放大境框注塑模结构和工作原理。该模具一模两腔,矩形截面侧浇口进浇,收缩型芯内抽芯和旋转型芯脱螺纹。成型塑件孔壁环形凹槽的型芯由6个大扇瓣块和6个小扇瓣块组合而成,通过大、小扇瓣块相对运动,使12个扇瓣块径向内移,实现环形凹槽抽芯。同时利用开模力,通过齿轮-齿条传动,使横向型芯旋转脱螺纹。     

沟槽参数对螺旋槽液膜密封空化诱发影响分析

为进一步探索液膜密封端面空穴发生诱因及其影响规律,本文基于JFO（Jakobsson-Floberg-Olsson）空化边界,建立双坝区中间开螺旋槽的液膜密封物理模型,通过空穴可视化实验验证了程序算法的准确性,分析了不同空化边界下液膜密封的空穴特征。以密封面间润滑液体的液膜压力和密度变化为判据,探讨了槽深、槽角和槽数等参数对液膜始破位置、重生位置及空穴发生面积的影响。结果表明：相比Half-Sommerfeld和Reynolds空化边界,基于JFO空化边界计算的空穴特征能更好地与实验结果相吻合;较大槽深和槽数,均缩小了液膜始破位置沿螺旋方向的空穴长度和液膜重生位置沿周向的空穴宽度,前者提升了两位置的空穴度,而后者降低了液膜重生位置的空穴度;槽角的增大,在扩大两位置空穴尺度的同时,提升了相应位置的空穴度;而液膜中空穴发生面积随槽深的增加线性降低,在较小槽角时先线性增加后随槽角增大呈抛物线增加趋势,而在槽数大于12时随槽数增加线性降低。