Design Of Such Probes Research Articles

Insight into the spatial interaction of D-π-A bridge derived cyanines and nitroreductase for fluorescent cancer hypoxia detection

Nitroreductase (NTR) detection in tumor is critical because NTR level is correlated with hypoxia degree and cancer prognosis. With the feature of high sensitivity and selectivity, fluorescence organic probes for NTR detection exhibited a promising future for tumor hypoxia detection. However, the discovery and design of such probes have been impeded due to the lack of the understanding of spatial match and mismatch of these probes with NTR. Here, we have developed two new nitrophenyl-functionalized trimethincyanine (Cy3) probes with para- or meta- positions of nitro-group in phenyl ring. Para-nitrophenyl substituted Cy3 (pNP-Cy3) exhibited a remarkable response to NTR (20-fold fluorescence enhancement) with good selectivity and sensitivity. Experimental and theoretical analysis verified that the substituent position of nitro group on phenyl ring of dyes altered the spatial arrangement of nitro-substituent group, thereby modulated the spatial match and mismatch between Cy3 dyes and binding domain of NTR, and consequently led to a different fluorescent turn-on response. In tumor-bearing mice model, hypoxia status of A549 xenografted tumor of mice was successfully delineated by using pNP-Cy3. These results may provide a clue for designing new cyanine-derived NTR probe to monitor NTR-overexpressed hypoxia cancer cells.

One-Component DNA Mechanoprobes for Facile Mechanosensing in Photopolymerized Hydrogels and Elastomers

DNA mechanosensors offer unique properties for mechano-adaptive and self-reporting materials, such as programmable bond strength and geometrical strain response, tunable fluorescent strain sensing, interfacing to biological systems, and the ability to store mechanical information. However, the facile incorporation of advanced DNA motifs into polymer networks and achieving robustness in application settings remain difficult. Herein, we introduce one-component DNA mechanoprobes that can be easily polymerized into polymer hydrogels and even elastomers to allow strain-induced fluorescence sensing. The all-in-one mechanoprobe contains a DNA hairpin for programmable force sensing, an internal fluorophore-quencher pair as a reporter, and methacrylamide groups on both ends for rapid and facile photopolymerization into networks based on the nontoxic water-soluble monomer methoxy triethylene glycol acrylate (mTEGA). In addition to mechanosensing hydrogels, we utilize the low Tg of p(mTEGA) to develop the first bulk elastomer materials with DNA force sensors, which show high elasticity and stronger mechanofluorescence. The system makes decisive steps forward for DNA-based mechanoprobes by overcoming the classical multicomponent design of such probes, allowing photopolymerization useful for the design of complex objects or even 3D printing and demonstrating that such motifs may even be useful in dry bulk materials.

OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes

Oligonucleotide (oligo)-based FISH has emerged as an important tool for the study of chromosome organization and gene expression and has been empowered by the commercial availability of highly complex pools of oligos. However, a dedicated bioinformatic design utility has yet to be created specifically for the purpose of identifying optimal oligo FISH probe sequences on the genome-wide scale. Here, we introduce OligoMiner, a rapid and robust computational pipeline for the genome-scale design of oligo FISH probes that affords the scientist exact control over the parameters of each probe. Our streamlined method uses standard bioinformatic file formats, allowing users to seamlessly integrate new and existing utilities into the pipeline as desired, and introduces a method for evaluating the specificity of each probe molecule that connects simulated hybridization energetics to rapidly generated sequence alignments using supervised machine learning. We demonstrate the scalability of our approach by performing genome-scale probe discovery in numerous model organism genomes and showcase the performance of the resulting probes with diffraction-limited and single-molecule superresolution imaging of chromosomal and RNA targets. We anticipate that this pipeline will make the FISH probe design process much more accessible and will more broadly facilitate the design of pools of hybridization probes for a variety of applications.

Computational study and error analysis of an integrated sampling-probe and gas-analyzer for mixing measurements in supersonic flow

Concentration probes are employed in supersonic flow mixing measurements. Because the typical design of such probes is essentially based on an inviscid, adiabatic, quasi-1D analysis, the scope of this work is to understand better and quantify the severe impact of viscous effects on the probe’s internal gasdynamics and the associated uncertainties in the measured quantities via a computational fluid dynamics analysis. Specifically, the focus is on the augmented errors due to the aforementioned viscous effects when coupled with various cases of probe-flow misalignment, which is a typical scenario encountered in mixing measurements of binary gas compositions (air and helium in the present work) in vortex-dominated flows. Results show phenomena such as shock induced boundary layer separation and the formation of an oblique shock train. These flow features are found to noticeably affect the accuracy of the composition measurement. The errors associated with the inviscid, adiabatic, quasi-1D analysis of the probes are quantified in this study.

Selection of COMBO-FISH Probes for Multi-Purpose Applications

The common method of fluorescence in situ hybridisation (FISH) is based on labelling DNA by the use of single stranded DNA-probes with appropriate markers binding to the counterpart within the genome (6). Originally FISH was performed with DNA biochemical amplified either in yeast (Yeast Artificial Chromosome, YAC) or in bacteria (Bacterial Artificial Chromosome, BAC). To achieve DNA hybridisation using these probes is requiring thermal and chemical treatment for denaturation after fixation of the specimen (7,8). These treatments together with the relatively large amount of probe DNA added, lead to several changes of the three dimensional nuclear structure resulting in a poor reproduction of the chromosomal and chromatin arrangement (9). Furthermore the creation of those probes does not require any knowledge about the exact sequence of the desired genomic region but only about the specific enzymatic cutting locations within the genome. Thus the length of the labelling probes is determined by this enzymatic and chemical processes rather than by the target sequence specificity of the markers. Nevertheless, FISH is still a standard method (6) used with great success in various fields in research and diagnostics and has fundamental impact on nowadays medical care. As mentioned, the conventional FISH method has at least two major disadvantages, on the one hand the destruction of the three dimensional structure due to denaturation treatment, which is a general problem for research in the field of genomic alterations and evaluation of the 3D-chromatin structure, and on the other hand the fully or correctly targeting labelling which is already a problem for labelling probe production itself. Both drawbacks can be overcome by the combinatorial design and usage of short oligonucleotide sequences which bind specifically to the desired genomic sequence. The design of such probes has become possible since the human genome has totally been sequenced and since there are methods of automated synthesising pre-determined DNA sequences industrially. The problem of conserving the original chromosomal structure can be solved by using triplex forming oligonucleotides (TFOs) of a length between 15 and 30 bases, that bind as a third strand into the major groove of the double stranded DNA within the nucleus. The usage of TFOs avoids denaturation of the specimen and in this way conserves the native structure of the chromatin. On the other hand such short probes lack uniqueness within the genome. This problem is solved by the usage of combinatorial sets of short oligonucleotides which only cover the selected region but do not co-localise anywhere else in the genome. This method is called Combinatorial Oligo Fluorescence In Situ Hybridisation (COMBO-FISH) (10). It can be used as well with the described TFOs as with common double-strand forming probes (Watson-Crick binding probes) both composed of DNA or peptide nucleic acids (PNAs) (10,11,12). Schmitt et al. have described the algorithms for designing such TFO-probes in ref. (12). By now this algorithm can also be used to identify and design Watson-Crick-binding probes (13). In the course of these algorithms one has to find the first and the last nucleotide of the genetic region of interest (ROI) first. With this numbers a set of oligonucleotides binding to this genomic ROI with a minimum of accessory binding sites and no other co-localisation site within the genome is computed. As parameters for this search a minimum and a maximum length of the oligonucleotides, the number of probes determined and the maximum cluster size of probes which are allowed to bind within a sequence of selectable length outside the labelling site are to be defined. The algorithms Schmitt et al. (12) created can be used as well on the genomic sequence database of the human genome as provided by the NCBI (14) as on a self-created database including the

Flow towards a guarded sampling probe: Modelling of a 2D flow cell

Analytic and numerical methods are used to predict fluid flow in a thin rectangular sheet of porous material when fluid is extracted through a port at the midpoint of one of the edges of the sheet. The aim is to model (in 2 dimensions) flow towards a sampling probe used to extract reservoir fluids from the rock surrounding a newly-drilled well. Such samples are usually contaminated with drilling fluid filtrate that saturates the rock adjacent to the wellbore, but a guarded probe can eliminate this contamination. Complex variable techniques are used to determine the flow of incompressible fluids when the interface between the two fluids is sharp, and filtrate and original pore fluid have identical viscosities. Results are compared against the predictions of a numerical reservoir simulator, and agreement is reasonable once numerical dispersion has been accounted for. Numerical results are also obtained for the case when the viscosities of the two fluids differ. The results give confidence in the use of 3-dimensional numerical computations to predict flow towards a guarded probe in a real wellbore, and thereby to optimize the design of such probes.

Probing the Active Site of a Diels−Alderase Ribozyme by Photoaffinity Cross-Linking

The active site of a Diels-Alderase ribozyme is located in solution by photoaffinity cross-linking using a productlike azidobenzyl probe. Two key nucleotides are identified that contact the Diels-Alder product in a conformation-dependent fashion. The design of such probes does not require knowledge of the three-dimensional structure of the ribozyme, and the technique yields both static and dynamic structural information. This work establishes photoaffinity cross-linking as an empirical approach that is applied here for the first time to an artificial ribozyme.

Fluorescent and photoactivable probes in depth-dependent analysis of membranes

This report summarizes our efforts towards depth-dependent analysis of membranes by design of suitable fluorescent and photoactivable lipid probes, which can be incorporated into membranes. The objective of depth-dependent analysis has been two fold, one to obtain information on lipid domains and other on transmembrane domains of membrane-bound proteins. In view of increasing importance of lipid rafts and other localized domain and limited success in case of structure determination of membrane-bound proteins vis-à-vis their soluble counterparts, it is tempting to rapidly attach fluorescent or photoactivable probes to lipids to get a probes where relatively little attention is paid to design of such probes. We have shown here how careful design of such probes is required to immobilize such probes in membranes for effective depth-dependent analysis of membranes. An effective design has become important when identification of putative transmembrane domains predicted primarily from the genome data based on hydropathy plots, often needs confirmation by contemporary methodology.

Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for fluorescence in situ hybridization is a result of ribosomal higher order structure.

The use of 16S rRNA targeted gene probes for the direct analysis of microbial communities has revolutionized the field of microbial ecology, yet a comprehensive approach for the design of such probes does not exist. The development of 16S rRNA targeted oligonucleotide probes for use with fluorescence in situ hybridization (FISH) procedures has been especially difficult as a result of the complex nature of the rRNA target molecule. In this study a systematic comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to determine if target location influences the hybridization efficiency of oligonucleotide probes when used with in situ hybridization protocols for the detection of whole microbial cells. Five unique universal 12-mer oligonucleotide sequences, located at different regions of the 16S rRNA molecule, were identified by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA sequences. The complements of these oligomeric sequences were chemically synthesized for use as probes and end labeled with either [gamma-32P]ATP or the fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for each of the probes was determined by hybridization to heat-denatured RNA immobilized on blots or to formaldehyde fixed whole cells. All of the probes hybridized with equal efficiency to denatured RNA. However, the probes exhibited a wide range of sensitivity (from none to very strong) when hybridized with whole cells using a previously developed FISH procedure. Differential hybridization efficiencies against whole cells could not be attributed to cell wall type, since the relative probe efficiency was preserved when either Gram-negative or -positive cells were used. These studies represent one of the first attempts to systematically define criteria for 16S rRNA targeted probe design for use against whole cells and establish target site location as a critical parameter in probe design.

Recent developments in streptococcal taxonomy

The classification of the streptococci has changed considerably in recent years, with the description of several new species, the re-naming of others, and the re-allocation of some former Streptococcus species to different genera. Phenotypic identification schemes have not kept pace with these taxonomic developments and there is considerable scope for improvement, perhaps with increased use of rapid enzyme-based methods involving fluorogenic or chromogenic substrates. Nucleic acid probes have been described for identification of several streptococcal species and the design of such probes has been greatly facilitated by the availability of 16S rRNA sequence data for most species. The pathogenic significance of some of the more recently described species is not fully understood. Many, if not all, of the oral streptococci appear to be involved in infective endocarditis, although not with equal frequency; several can also be a significant cause of infection in immunologically-compromised patients. Within the oral streptococci the ‘Streptococcus milleri group’ are frequently isolated from purulent infections and there is evidence for some specificity in the distribution of Streptococcus intermedius, S. constellatus and S. anginosus at different body sites. The S. mutans group are important aetiological agents in dental caries, and almost any of the oral streptococci may be involved in other infections in the mouth. In addition to the obvious need for more epidemiological data on the role of the newly described species in human and animal disease, further studies on their virulence mechanisms are also indicated.

Determination of Absolute Sound Levels and Acoustic Absorption Coefficients by Thermocouple Probes—Experiment

A stable, readily constructable thermocouple probe has been developed for determining absolute sound levels in ultrasonic fields in liquid media. This paper includes criteria for design of such probes and a discussion of experimental measurements made with such a device. It is a consequence of the method that if the sound intensity at an appropriate size thermocouple junction imbedded in an absorbing medium is known the acoustic absorption coefficient of the material can be determined. The method thus makes possible the determination of absorption coefficients of minute quantities of material. The probe consists of a thermocouple imbedded in a sound absorbing medium which closely matches in density and sound velocity the medium in which the sound level is to be determined. In use the transducer which generates the acoustic field is exctied to generate sound pulses with a rectangular envelope. The initial time rate of change of the temperature at the thermocouple junction is determined. In addition to the measurement of the temperature change, the calculation of the absolute sound intensity requires only a knowledge of the absorption coefficient of the imbedding material and its heat capacity per unit volume at the temperature at which the measurements are made. The experimental results include a comparison of the sound level determined by a thermocouple probe and a determination by radiation pressure methods. The values obtained by the two methods agree within the uncertainty of the experimental measurements.