Activatable fluorescent probes in fluorescence-guided surgery: Practical considerations

Fluorescence-guided imaging during surgery is a promising technique that is increasingly used to aid surgeons in identifying sites of tumor and surgical margins. Of the two types of fluorescent probes, always-on and activatable, activatable probes are preferred because they produce higher target-to-background ratios, thus improving sensitivity compared with always-on probes that must contend with considerable background signal. There are two types of activatable probes: 1) enzyme-reactive probes that are normally quenched but can be activated after cleavage by cancer-specific enzymes (activity-based probes) and 2) molecular-binding probes which use cancer targeting moieties such as monoclonal antibodies to target receptors found in abundance on cancers and are activated after internalization and lysosomal processing (binding-based probes). For fluorescence-guided intraoperative surgery, enzyme-reactive probes are superior because they can react quickly, require smaller dosages especially for topical applications, have limited side effects, and have favorable pharmacokinetics. Enzyme-reactive probes are easier to use, fit better into existing work flows in the operating room and have minimal toxicity. Although difficult to prove, it is assumed that the guidance provided to surgeons by these probes results in more effective surgeries with better outcomes for patients. In this review, we compare these two types of activatable fluorescent probes for their ease of use and efficacy.     

Fluorescence Characteristics of Hydrophobic Partial Agonist Probes of the Cholecystokinin Receptor

Fluorescence spectroscopic studies are powerful tools for the evaluation of receptor structure and the dynamic changes associated with receptor activation. Here, we have developed two chemically distinct fluorescent probes of the cholecystokinin (CCK) receptor by attaching acrylodan or a nitrobenzoxadiazole moiety to the amino terminus of a partial agonist CCK analogue. These two probes were able to bind to the CCK receptor specifically and with high affinity, and were able to elicit only submaximal intracellular calcium responses typical of partial agonists. The fluorescence characteristics of these probes were compared with those previously reported for structurally-related full agonist and antagonist probes. Like the previous probes, the partial agonist probes exhibited longer fluorescence lifetimes and increased anisotropy when bound to the receptor than when free in solution. The receptor-bound probes were not easily quenched by potassium iodide, suggesting that the fluorophores were protected from the extracellular aqueous milieu. The fluorescence characteristics of the partial agonist probes were quite similar to those of the analogous full agonist probes and quite distinct from the analogous antagonist probes. These data suggest that the partially activated conformational state of this receptor is more closely related to its fully active state than to its inactive state.     

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.     

Design, synthesis, modeling, biological evaluation and photoaffinity labeling studies of novel series of photoreactive benzamide probes for histone deacetylase 2

The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs.     

Use of oligodeoxynucleotide signature probes for identification of physiological groups of methylotrophic bacteria

Oligodeoxynucleotide sequences that uniquely complemented 16S rRNAs of each group of methylotrophs were synthesized and used as hybridization probes for the identification of methylotrophic bacteria possessing the serine and ribulose monophosphate (RuMP) pathways for formaldehyde fixation. The specificity of the probes was determined by hybridizing radiolabeled probes with slot-blotted RNAs of methylotrophs and other eubacteria followed by autoradiography. The washing temperature was determined experimentally to be 50 and 52 degrees C for 9-alpha (serine pathway) and 10-gamma (RuMP pathway) probes, respectively. RNAs isolated from serine pathway methylotrophs bound to probe 9-alpha, and RNAs from RuMP pathway methylotrophs bound to probe 10-gamma. Nonmethylotrophic eubacterial RNAs did not bind to either probe. The probes were also labeled with fluorescent dyes. Cells fixed to microscope slides were hybridized with these probes, washed, and examined in a fluorescence microscope equipped with appropriate filter sets. Cells of methylotrophic bacteria possessing the serine or RuMP pathway specifically bind probes designed for each group. Samples with a mixture of cells of type I and II methanotrophs were detected and differentiated with single probes or mixed probes labeled with different fluorescent dyes, which enabled the detection of both types of cells in the same microscopic field.     

Use of oligodeoxynucleotide signature probes for identification of physiological groups of methylotrophic bacteria.

Oligodeoxynucleotide sequences that uniquely complemented 16S rRNAs of each group of methylotrophs were synthesized and used as hybridization probes for the identification of methylotrophic bacteria possessing the serine and ribulose monophosphate (RuMP) pathways for formaldehyde fixation. The specificity of the probes was determined by hybridizing radiolabeled probes with slot-blotted RNAs of methylotrophs and other eubacteria followed by autoradiography. The washing temperature was determined experimentally to be 50 and 52 degrees C for 9-alpha (serine pathway) and 10-gamma (RuMP pathway) probes, respectively. RNAs isolated from serine pathway methylotrophs bound to probe 9-alpha, and RNAs from RuMP pathway methylotrophs bound to probe 10-gamma. Nonmethylotrophic eubacterial RNAs did not bind to either probe. The probes were also labeled with fluorescent dyes. Cells fixed to microscope slides were hybridized with these probes, washed, and examined in a fluorescence microscope equipped with appropriate filter sets. Cells of methylotrophic bacteria possessing the serine or RuMP pathway specifically bind probes designed for each group. Samples with a mixture of cells of type I and II methanotrophs were detected and differentiated with single probes or mixed probes labeled with different fluorescent dyes, which enabled the detection of both types of cells in the same microscopic field.     

Application of rRNA-based probes for observing marine nanoplanktonic protists.

The use of small-subunit rRNA-based oligonucleotides as probes for detecting marine nanoplanktonic protists was examined with a ciliate (an Uronema sp.), a flagellate (a Cafeteria sp.), and mixed assemblages of protists from enrichment cultures and natural seawater samples. Flow cytometry and epifluorescence microscopy analyses demonstrated that hybridizations employing fluorescein-labeled, eukaryote-specific probes intensely stained logarithmically growing protists, whereas these same protist strains in late stationary growth were barely detectable. The fluorescence intensity due to probe binding was significantly enhanced by the use of probes end labeled with biotin, which were detected by fluorescein-labeled avidin. The degree of signal amplification ranged from two- to fivefold for cultured protists in both logarithmic and stationary growth phases. Mixed assemblages of heterotrophic protists from enrichment cultures were also intensely labeled by rRNA-targeted oligonucleotide probes by the biotin-avidin detection system. Protists in late stationary growth phase and natural assemblages of protists that were otherwise undetectable when hybridized with fluorescein-labeled probes were easily visualized by this approach. In the latter samples, hybridization with multiple, biotin-labeled probes was necessary for detection of naturally occurring marine protists by epifluorescence microscopy. The signal amplification obtained with the biotin-avidin system should increase the utility of rRNA-targeted probes for identifying protists and facilitate characterization of the population structure and distribution of protists in aquatic environments.     

等Tm检测探针寡核苷酸芯片在p53突变热点检测中的应用

用等长探针检测基因的点突变,不同GC含量探针的碱基错分辨率很难均一。尝试利用探针近似等Tm的原则设计、制备了检测抑癌基因p53外显子7中密码子245、248、249单碱基突变及缺失的寡核苷酸芯片。实验得到较好的碱基错配分辨率,检测不同位点的碱基错配分辨率较为一致,芯片检测结果与测序结果一致。实验结果为制备检测p53常见热点突变的寡核酸芯片奠定了基础。     

Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies.

Universal oligonucleotide hybridization probes targeting the small-subunit rRNA are commonly used to quantify total microbial representation in environmental samples. Universal probes also serve to normalize results obtained with probes targeting specific phylogenetic groups of microorganisms. In this study, six universal probes were evaluated for stability of probe-target duplexes by using rRNA from nine organisms representing the three domains of Bacteria, Archaea, and Eucarya. Domain-specific variations in dissociation temperatures were observed for all probes. This could lead to a significant bias when these probes are used to quantify microbial populations in environmental samples. We suggest lowering the posthybridization wash stringency for two of the universal probes (S-*-Univ-1390-a-A-18 and S-*-Univ-1392-a-A-15) examined. These two probes were evaluated with traditional and modified hybridization conditions to characterize defined mixtures of rRNAs extracted from pure cultures and rRNA samples obtained from anaerobic digester samples. Probe S-*-Univ-1390-a-A-18 provided excellent estimations of domain-level community composition of these samples and is recommended for future use in microbial ecology studies.     

Advanced functional fluorescent probes for cell plasma membranes

Imaging the plasma membrane (PM) by fluorescence techniques using molecular fluorescent probes enable cell segmentation, studying membrane organization and dynamics, formation, and tracking of vesicles. Rational molecular design brings fluorescent PM probes to a new level, providing PM probes with new functions beyond basic PM staining and imaging. We herein review the latest advances in fluorescent PM probes for chemical and biophysical sensing as well as for super-resolution imaging.     

Improved in situ hybridization efficiency with locked-nucleic-acid-incorporated DNA probes

Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.     

A new method for rapid screening of bacterial species- or subspecies-specific DNA probes

A simple assay for the rapid screening of bacterial species- or subspecies-specific DNA probes for the random cloning method is presented, involving the use of genomic DNAs as probes and recombinant plasmid DNAs containing genomic DNA digested with HindIII as targets. The optimal amount of target DNAs and the concentration of digoxigenin-labeled genomic DNA probes were 20 ng and 100 ng ml(-1) (or 10 ng and 200 ng ml(-1)), respectively. The method was applied to the development of Fusobacterium nucleatum subspecies-specific probes. Our results showed that four out of 96 probes were F. nucleatum subspecies-specific, which was confirmed by Southern blot analysis. Our results indicate that the new method can be used for the rapid screening of species- or subspecies-specific probes.     

Comparison of different kinds of probes used for analysis of variant telomeric sequences

In this work we aimed to compare and critically evaluate results obtained by different types of probes used for hybridisation to detect variant telomeric sequences with respect to their reliability and information value. Using slot-blot hybridisation we investigated three types of probes (oligonucleotides, cloned fragments and concatenated probes) under various conditions of hybridisation and washing. The concatenated probes exhibited the highest specificity although all three types are suitable for hybridisation of telomeric sequences under appropriate experimental conditions. We demonstrate how understanding generated from these data enables interpretation of hybridisation patterns of oligonucleotide probes to genomic DNAs.     

Screening of a unique lectin from 16 cultivable mushrooms with hybrid glycoprotein and neoproteoglycan probes and purification of a novel N-acetylglucosamine-specific lectin from Oudemansiella platyphylla fruiting body

Hybrid glycoprotein and neoproteoglycan probes were prepared by coupling various glycoproteins or polysaccharides to peroxidase or biotinyl bovine serum albumin, respectively. Lectins recognizable by the neoglycoconjugate probes were extracted from 16 cultivable mushrooms. Dot-blot assay revealed five extracts to be reactive with only hybrid glycoprotein probes, but others also reacted with neoproteoglycan probes. According to the reactivity pattern with probe screening, the one lectin from Oudemansiella platyphylla extract (OPL) bound best with asialotransferrin-- and asialoagalactotransferrin--peroxidase probes and was isolated using an asialotransferrin column, but it did not bind with other hybrid glycoprotein or neoproteoglycan probes. OPL, consisting of two polypeptides with high homology in the N-terminal amino acid sequences, exhibited weak hemagglutinating activity. Purified OPL specifically bound the beta-GlcNAc probe among various biotinylated polymeric sugar probes, while it exhibited essentially the same binding specificity toward neoglycoconjugate probes as that of the crude extract, showing a preference for the asialobiantennary complex type of N-linked glycans. These results indicate that the neoglycoconjugate probes are valuable in lectin screening.     

Two-dimensional microelectrophoresis in supported lipid bilayers

We report the application of supported bilayers for two-dimensional microelectrophoresis. This method allows the lateral separation and accumulation of charged amphiphilic molecular probes in bilayers by application of an electric field parallel to the bilayer surface. Diffusion coefficient and mobility of the fluorescent probes are determined by observation of the fluorescence recovery after photobleaching (pattern bleaching). The diffusion coefficients and the mobilities of oppositely charged fluorescent probes in one bilayer can be determined independently from a single measurement. By analysis of the motion of charged and uncharged probes in one membrane one can distinguish between the motion caused by the electric field acting on the charge of individual probes and that caused by frictional forces due to electroosmosis.     

Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes

Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.