Design of base-discriminating fluorescent nucleoside and its application to t/c SNP typing

We report a novel method for base detection using a base-discriminating fluorescent (BDF) nucleoside. We developed BDF probes containing methoxybenzodeazaadenine MDA and methoxybenzodeazainosine MDI, which give strong fluorescence only when the base on the complementary strand is cytosine and thymine, respectively. Thus, the MDA- and MDI-containing ODNs can be used as a very effective BDF probe for the detection of single base alterations, such as SNPs and point mutations. The present method using BDF probes is a very powerful tool for SNP typing that does not require any enzymes and time-consuming steps, and can avoid hybridization errors. In addition, a combination of MDA- and MDI-containing BDF probes facilitates the T/C SNP typing of a heterozygous sample.     

Synthesis and properties of novel base-discriminating fluorescent (BDF) nucleosides: a highly polarity-sensitive fluorophore for SNP typing

We have developed novel alkanoylpyrene-labeled BDF nucleosides, ^AMPyU and ^MPyU. These nucleosides exhibit strong fluorescence emission at long wavelength that is highly sensitive to solvent polarity. BDF probes containing ^AMPyU selectively emit fluorescence only when the base opposite BDF nucleoside is adenine and act as effective reporter probes for homogeneous SNP typing.     

ResonSense: simple linear fluorescent probes for quantitative homogeneous rapid polymerase chain reaction

Here we report a strand-specific fluorescent homogeneous assay format for rapid polymerase chain reaction (PCR). A number of similar assays are commonly used for research applications and are an ideal solution for a closed tube quantitative PCR. These assays use fluorescent resonant energy transfer (FRET) between donor and acceptor fluorescent moieties as the reporting mechanism. However, for different reasons these assays do not report amplification when very rapid cycling times are used. This is because current assays, such as TaqMan^®, are limited, in terms of assay speed, by the 5′-3′ exonuclease activity of Taq DNA polymerase. Other assays based on hybridisation require either a complex de-conformational event to occur, or require more than one probe to report amplification. Reducing the complexity of the experiment reduces costs in terms of design, optimisation and manufacture. Here, we describe ResonSense^® chemistries that use a simple linear fluorescent-labelled probe and a DNA minor-groove binding dye as either donor or acceptor moieties in a homogeneous assay format on the LightCycler^®. This assay format will provide for rapid analysis of samples and so it is particularly well suited to point-of-use testing.     

Copper(II)-quenched oligonucleotide probes for fluorescent DNA sensing

A copper(II)-quenched molecular beacon was prepared by attaching fluorescein to the 3'-end and a copper(II) complex to the 5'-end of DNA. In the presence of complementary DNA, copper(II) and dye are spatially separated in the duplex and fluorescence increases up to 15-fold, with excellent discrimination of single base mismatches.     

Fluorescence-labelled DNA probes to detect complementary sequences in homogeneous media

Sensitive, safe and easy-to-use probes for the detection of nucleic acids are urgently called for. To this end we are in the process of developing a fluorescence-based technique to work in homogeneous assay media. We have examined pyrene and fluorescein as fluorescent labels for natural DNA probes. A fraction of the cytosine residues of a single-stranded cDNA was randomly labelled with either pyrene or fluorescein using the bisulfite-catalyzed diamine reaction. Both fluorophores showed fluorescence quenching when the labelled probe was hybridized with its complementary strand and we describe the changes in steady-state fluorescence intensity that occurred upon hybridization. Our results demonstrate that pyrene quenching is more efficient than fluorescein quenching and thus pyrene-labelled probes are more sensitive for detecting and quantifying DNA from natural sources.     

Pyrene-labeled deoxyuridine and deoxyadenosine: fluorescent discriminating phenomena in their oligonucleotides

Pyrene-labeled deoxyuridine (U^P) and deoxyadenosine (A^P) units, which are substituted at the C-5 and C-8 positions, respectively, are fluorescent unnatural nucleosides. When duplexes are formed, these nucleobases feature strong and stable interstrand stacking interactions between the two pyrene units, which compensate for the loss of hydrogen bonding and induce characteristic pyrene excimer emissions.     

Sequence specific interstrand photocrosslinking for effective SNP typing

We describe a simple and inexpensive SNP typing method by using sequence specific interstrand photocrosslinking via p-carbamoylvinyl phenol nucleosides. Interstrand photocrosslinking showed a high degree of single nucleotide specificity as high as 10(3)-fold and more, and can be used in the diagnostic detection of DNA sequences.     

New fluorescent probes for carbonic anhydrases

We report the synthesis and fluorescence properties of naphthalenesulfonamide derivatives as active site probes for carbonic anhydrases.     

氨肽酶N荧光探针的研究进展

氨肽酶N(aminopeptidase N,APN)是一种外肽酶,广泛存在于哺乳动物体内,可从蛋白质多肽链的N末端水解中性或碱性氨基酸,在人体中具有多种重要的生理功能。APN可作为癌症诊断的标志物,尿液中APN也可作为肾小球肾炎的早期生物标志物。本文综述了APN荧光探针的研究进展,主要包括亲和力型APN荧光探针和反应型APN荧光探针,并对它们的优缺点进行了比较;最后对APN荧光探针的发展前景进行了展望。     

DNA hole transport on an electrode: application to effective photoelectrochemical SNP typing

A useful feature of DNA is that long-range hole transport through DNA is readily achieved. Photostimulated long-range hole transport through DNA has prospective use in the development of a conceptually new electrochemical single-nucleotide polymorphism (SNP) typing method for use as a versatile platform for gene diagnostics and pharmacogenetics. We have applied artificial DNAs designed for photostimulated long-range hole transport through DNA to SNP typing. By hybridizing photosensitizer-equipped DNA probes, immobilized on gold working electrodes, with a target DNA strand containing an SNP site, we observed a cathodic photocurrent, which markedly changed depending on the nature of the base at the specific site. The use of a combination of hole-transporting bases constitutes a very powerful method for a single-step electrochemical assay applicable to SNP typing of all types of sequences.     

荧光素类荧光分子探针

荧光素衍生物是重要的荧光探针,在检测和生物成像等领域中显示出巨大的前景。因此,急需对功能性荧光素结构探针的设计策略进行深入研究。通常通过引入醛基或酯化到荧光素呫吨环和苯部分来构建探针,由于其高活性,这些衍生物可以与分析物复合以发生颜色和荧光强度的变化。本文总结了荧光素的修饰位点及方法,介绍了荧光素探针的合成、性质及应用,并对近五年荧光素探针对不同分析物(包括金属阳离子、阴离子、小分子和生物大分子)的检测进行分类说明,旨在为高灵敏度荧光素探针的筛选和生物检测提供参考,并推动其在分析物传感和检测中的进一步应用。     

Bifunctionalised long-wavelength fluorescent probes for biological applications

The synthesis of benzo[a]phenoxazinium chlorides which are bifunctionalised in position 2 with 4-ethoxy-4-oxobutoxyl, 3-hydroxypropoxyl or 3-chloropropoxyl groups, and in position 9 with the (aminopropyl)amino group, was efficiently performed. The covalent labelling of valine was carried out by using one of the new fluorophores obtained. Photophysical studies in the homogeneous media of ethanol, distilled water and simulated physiological conditions revealed that all the compounds absorbed and emitted from 610 to 651 nm.     

Biomarker-targeted fluorescent probes for breast cancer imaging

This review summarized fluorescent probes for breast cancer imaging according to different biomarkers probes recognized.     

Reaction based fluorescent probes for hydrogen sulfide

A reaction based fluorescence turn-on strategy for hydrogen sulfide (H(2)S) was developed. This strategy was based on a H(2)S-specific Michael addition-cyclization sequence. Other biological thiols such as cysteine and glutathione did not pursue the reaction and therefore did not turn on the fluorescence/consume the substrates. The probes showed good selectivity and sensitivity for hydrogen sulfide.     

银纳米簇荧光探针检测L-半胱氨酸

常温下合成了二氢硫辛酸(DHLA)包裹的银纳米簇(AgNCs),并基于L-半胱氨酸对AgNCs的荧光猝灭现象构建了AgNCs荧光探针对Cys的检测方法。结果表明,在优化条件下,AgNCs的荧光猝灭程度和Cys浓度在2.0~100μmol/L范围内呈现良好的线性关系(R2=0.998),检出限为1.77μmol/L(S/N=3)。在人体血清样品中Cys检测的加标回收率为94.0%~102.4%。     

Organelle targetable fluorescent probes for hydrogen peroxide

The review summarized organelle target strategies, structures, fluorescence behavior and biological applications of H₂O₂ fluorescent probes with mitochondria, nuclei and lysosomes-targetable ability.     

Organelle targetable fluorescent probes for hydrogen peroxide

Hydrogen peroxide(H_2 O_2), as important products of oxygen metabolism, plays an important role in many biological processes, such as immune responses and cellular signal transduction. However, abnormal production of H_2 O_2 can damage cellular biomolecules, which was closely associated with many diseases.Thus, it is urgent to monitor the level change of H_2 O_2 in living cells, particularly at subcellular levels.Toward this end, a wide variety of H_2 O_2 fluorescent probes have been designed, developed and applied for imaging of H_2 O_2 in subcellular levels. In this review, we highlight the representative cases of H_2 O_2 fluorescent probes with mitochondria, nuclei and lysosomes-targetable ability. The review contains organelle target strategies, structures, fluorescence behavior and biological applications of these probes.     

Triad base pairs containing fluorene unit for quencher-free SNP typing

We designed an A-selective fluorescent DNA probe, U^FL, which bears a 2-ethynylfluorene moiety covalently attached to the base dU, and incorporated it into a central position in the hairpin loop. We describe photophysical studies of triad base pairs containing an U^FL DNA probe and their application to single-nucleotide polymorphism (SNP) typing using novel quencher-free molecular beacons (MBs). The drastic changes in the fluorescence properties that arise upon changing the nature of the complementary base and FBs suggest that these triad base pairs are the key elements for quencher-free SNP typing.     

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。