Investigation of the binding profiles of the dasatinib-oligonucleotide conjugates (0.2, 1 and 5 nM) on Protein Microarrays
ABSTRACT: Using this approach the dasatinib-oligonucleotide conjugates was applied to planar arrays of >9,000 human proteins spotted in two technical replicates. All proteins in the commercially available ProtoArray® Human Protein MicroArray (Thermo Fisher Scientific) have been purified and arrayed under native conditions to allow such studies. We adopted this format for investigation of the binding profiles of the drug-oligonucleotide conjugates. Fluorophore-labeled drugs have previously been used to measure binding in protein arrays. The oligonucleotide-conjugated constructed allowed for locally amplified detection via RCA. Circularizing oligonucleotides (padlock probes) were designed with 5’ and 3’ ends complementary to adjacent segments of the oligonucleotides conjugated to the drug molecules. Once converted to oligonucleotide circles by ligation, the probes were replicated through localized RCA, primed by the drug-conjugated oligonucleotides, and visualized using fluorescence-labeled hybridization probes to the repeated sequence of the RCA products. RCA offers a signal enhancement of several hundredfold over singly fluorophore labeled compounds, permitting visualization of even single bound drug probes
Project description:Using this approach the dasatinib-oligonucleotide conjugates was applied to planar arrays of >9,000 human proteins spotted in two technical replicates. All proteins in the commercially available ProtoArray® Human Protein MicroArray (Thermo Fisher Scientific) have been purified and arrayed under native conditions to allow such studies. We adopted this format for investigation of the binding profiles of the drug-oligonucleotide conjugates. Fluorophore-labeled drugs have previously been used to measure binding in protein arrays. The oligonucleotide-conjugated constructed allowed for locally amplified detection via RCA. Circularizing oligonucleotides (padlock probes) were designed with 5’ and 3’ ends complementary to adjacent segments of the oligonucleotides conjugated to the drug molecules. Once converted to oligonucleotide circles by ligation, the probes were replicated through localized RCA, primed by the drug-conjugated oligonucleotides, and visualized using fluorescence-labeled hybridization probes to the repeated sequence of the RCA products. RCA offers a signal enhancement of several hundredfold over singly fluorophore labeled compounds, permitting visualization of even single bound drug probes
Project description:Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.
Project description:A novel colorimetric assay employing oligonucleotide-conjugated gold nanoparticle (AuNP probes) and rolling circle amplification (RCA) was developed for simple detection of mercuric ions (Hg2+). The thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry makes our detection system selective for Hg2+. In the presence of Hg2+, the thymine 12-mer oligonucleotide is unable to act as a primer for RCA due to the formation of T-Hg2+-T before the RCA reaction. However, in the absence of Hg2+, DNA coils as RCA products are generated during the RCA reaction, and is further labeled with AuNP probes. Colorimetric signals that depend on the amount of DNA coil-AuNP probe complexes were generated by drop-drying the reaction solution on nitrocellulose-based paper. As the reaction solution spread radially because of capillary action, the complexes formed a concentric red spot on the paper. The colorimetric signals of the red spots were rapidly measured with a portable spectrophotometer and determined as the ?E value, which indicates the calculated color intensity. Our assay displays great linearity (detection limit: 22.4 nM), precision, and reproducibility, thus demonstrating its utility for Hg2+ quantification in real samples. We suggest that our simple, portable, and cost-effective method could be used for on-site Hg2+ detections.
Project description:We describe a new, and vastly superior approach for labeling spherical nucleic acid conjugates (SNAs) with diagnostic probes. SNAs have been shown to provide the unique ability to traverse the cell membrane and deliver surface conjugated DNA into cells while preserving the DNA from nuclease degradation. Our previous work on preparing diagnostically labeled SNAs was labor intensive, relatively low yielding, and costly. Here, we describe a straightforward and facile preparation for labeling SNAs with optical and MR imaging probes with significantly improved physical properties. The synthesis of Gd(III) labeled DNA Au nanoparticle conjugates is achieved by sequential conjugation of 3'-thiol-modified oligonucleotides and cofunctionalization of the particle surface with the subsequent addition of 1,2 diothiolate modified chelates of Gd(III) (abbreviated: DNA-GdIII@AuNP). This new generation of SNA conjugates has a 2-fold increase of DNA labeling and a 1.4-fold increase in Gd(III) loading compared to published constructs. Furthermore, the relaxivity ( r1) is observed to increase 4.5-fold compared to the molecular dithiolane-Gd(III) complex, and 1.4-fold increase relative to previous particle constructs where the Gd(III) complexes were conjugated to the oligonucleotides rather than directly to the Au particle. Importantly, this simplified approach (2 steps) exploits the advantages of previous Gd(III) labeled SNA platforms; however, this new approach is scalable and eliminates modification of DNA for attaching the contrast agent, and the particles exhibit improved cell labeling.
Project description:Changes in cell identities and positions underlie tissue development and disease progression. Although single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of cell states, spatially resolved single-cell mapping presents a challenging task. We developed SCRINSHOT (Single-Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on murine and human organs. SCRINSHOT quantification of marker gene expression shows high correlation with published scRNA-Seq data over a broad range of gene expression levels. We demonstrate the utility of SCRINSHOT by mapping the locations of abundant and rare cell types along the murine airways. The amenability, multiplexity, and quantitative qualities of SCRINSHOT facilitate single-cell mRNA profiling of cell-state alterations in tissues under a variety of native and experimental conditions.
Project description:Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs) is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection. Interestingly, DNA mutations can be detected using fluorescently labeled oligonucleotide probes with only one fluorophore. This review summarizes recent research on single-labeled oligonucleotide probes that exhibit fluorescence changes after encountering target nucleic acids, such as guanine-quenching probes, cyanine-containing probes, probes containing a fluorophore-labeled base, and microenvironment-sensitive probes.
Project description:The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.
Project description:Fluorescence-based detection methods are being increasingly utilized in molecular analyses. Sequence-specific fluorescently-labeled probes are favored because they provide specific product identification. The most established fluorescence-based detection systems employ a resonance energy transfer mechanism effected through the interaction of two or more fluorophores or functional groups conjugated to oligonucleotide probes. The design, synthesis and purification of such multiple fluorophore-labeled probes can be technically challenging and expensive. By comparison, single fluorophore-labeled probes are easier to design and synthesize, and are straightforward to implement in molecular assays. We describe herein a novel fluorescent strategy for specific nucleic acid detection and genotyping. The format utilizes an internally quenched fluorescein-oligonucleotide conjugate that is subsequently dequenched following hybridization to the target with an attendant increase in fluorescence. Reversibility of the process with strand dissociation permits Tm-based assessment of bp complementarity and mismatches. Using this approach, we demonstrated specific detection, and discrimination of base substitutions of a variety of synthetic nucleic acid targets including Factor V Leiden and methylenetetrahydrofolate reductase. We further demonstrated compatibility of the novel chemistry with polymerase chain reaction by amplification and genotyping of the above listed loci and the human hemoglobin beta chain locus. In total, we analyzed 172 clinical samples, comprising wild-type, heterozygous and homozygous mutants of all three loci, with 100% accuracy as confirmed by DNA sequencing, established dual hybridization probe or high performance liquid chromatography-based methods. Our results indicate that the dequenching-based single fluorophore format is a feasible strategy for the specific detection of nucleic acids in solution, and that assays using this strategy can provide accurate genotyping results.
Project description:Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3' end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes.
Project description:Automated attachment of chemotherapeutic drugs to oligonucleotides through phosphoramidite chemistry and DNA synthesis has emerged as a powerful technology in constructing structure-defined and payload-tunable oligonucleotide-drug conjugates. In practice, however, in?vivo delivery of these oligonucleotides remains a challenge. Inspired by the systemic transport of hydrophobic payloads by serum albumin in nature, we report the development of a lipid-conjugated floxuridine homomeric oligonucleotide (LFU20) that "hitchhikes" with endogenous serum albumin for cancer chemotherapy. Upon intravenous injection, LFU20 immediately inserts into the hydrophobic cave of albumin to form an LFU20/albumin complex, which accumulates in the tumor by the enhanced permeability and retention (EPR) effect and internalizes into the lysosomes of cancer cells. After degradation, cytotoxic floxuridine monophosphate is released to inhibit cell proliferation.