Fluorescein-labeled Oligonucleotide Research Articles

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe.

The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen—alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 103 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.

Programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), and EBV-encoded RNA (EBER) expression in Hodgkin lymphoma.

Programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) are new targets in cancer immunotherapy. PD-1 protein is an immune checkpoint expressed in many tumors. Epstein-Barr virus (EBV) is present in malignant Hodgkin/Reed-Sternberg (HRS) cells in approximately 40-50% of Hodgkin lymphoma (HL). The aim of this study is to evaluate the clinical and prognostic importance of PD-1 and/or PD-L1 in HL and also to determine the association between EBV-encoded RNA (EBER) and PD-1/PD-L1. Formalin-fixed, paraffin-embedded tissue samples from 87 cases with HL were analyzed in this study. Immunohistochemical staining was performed to detect the PD-1 and PD-L1 expressions. Chromogenic in situ hybridization for EBER was performed using fluorescein-labeled oligonucleotide probes. PD-1 and PD-L1 expressions were found in 20% of the cases. The EBER positivity was found in 40 cases (45%). It has been found that co-expression of PD-1 and PD-L1 was associated with shorter survival although PD-1 or PD-L1 expressions were not found to be related with survival. Overall survival (OS) and disease-free survival (DFS) in cases without PD-1 and PD-L1 expressions were 135 and 107months, respectively. OS and DFS in cases with co-expression for PD-1 and PD-L1 were 24 and 20months, respectively, and these differences were found to be statistically significant for both OS and DFS (p = 0.002 and p = 0.003, respectively). Cox regression analysis showed that co-expression of PD-1 and PD-L1 was found to be an independent risk factor for prognosis (OR 6.9, 95% CI 1.9-24.3). Targeting PD-1 and/or PD-L1 is meaningful due to the 20% expression of each in HL, and we did not find an important association between PD-1 and PD-L1 and EBER expression in HL. Very poor outcome in cases with co-expression of PD-1/PD-L1 suggests new avenues to detect the new prognostic markers and also therapeutic approaches in HL.

Monomeric nature of dengue virus NS3 helicase and thermodynamic analysis of the interaction with single-stranded RNA.

Dengue virus nonstructural protein 3 (NS3) is a multifunctional protein formed by a superfamily-2 RNA helicase linked to a protease domain. In this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus NS3 helicase (NS3h) and to characterize fundamental properties of the interaction with single-stranded (ss)RNA. Pulsed field gradient-NMR spectroscopy was used to determine the effective hydrodynamic radius of NS3h, which was constant over a wide range of protein concentrations in the absence and presence of ssRNA. Size exclusion chromatography-static light scattering experiments showed that NS3h eluted as a monomeric molecule even in the presence of ssRNA. Binding of NS3h to ssRNA was studied by quantitative fluorescence titrations using fluorescein-labeled and unlabeled ssRNA oligonucleotides of different lengths, and the effect of the fluorescein label on the interaction parameters was also analyzed. Experimental results were well described by a statistical thermodynamic model based on the theory of non-specific interactions of large ligands to a one-dimensional lattice. We found that binding of NS3h to ssRNA oligonucleotides and to poly(A) is characterized by minimum and occluded binding site sizes both of 10 nucleotides and by a weak positive cooperativity between adjacent proteins.

Crystal Structures of the DNA-binding Domain Tetramer of the p53 Tumor Suppressor Family Member p73 Bound to Different Full-site Response Elements

How cells choose between developmental pathways remains a fundamental biological question. In the case of the p53 protein family, its three transcription factors (p73, p63, and p53) each trigger a gene expression pattern that leads to specific cellular pathways. At the same time, these transcription factors recognize the same response element (RE) consensus sequences, and their transactivation of target genes overlaps. We aimed to understand target gene selectivity at the molecular level by determining the crystal structures of the p73 DNA-binding domain (DBD) in complex with full-site REs that vary in sequence. We report two structures of the p73 DBD bound as a tetramer to 20-bp full-site REs based on two distinct quarter-sites: GAACA and GAACC. Our study confirms that the DNA-binding residues are conserved within the p53 family, whereas the dimerization and tetramerization interfaces diverge. Moreover, a conserved lysine residue in loop L1 of the DBD senses the presence of guanines in positions 2 and 3 of the quarter-site RE, whereas a conserved arginine in loop 3 adapts to changes in position 5. Sequence variations in the RE elicit a p73 conformational response that might explain target gene specificity.

Highly sensitive detection of superoxide dismutase based on an immunoassay with surface-enhanced fluorescence

Herein, a novel highly sensitive enhanced-fluorescence immunoassay for detection of superoxide dismutase (SOD) is established by combining surface-enhanced fluorescence (SEF) with immuno-magnetic separation. Based on a sandwich-type immunoassay, analytes in samples are first captured by magnetic beads coated with a monoclonal antibody and then "sandwiched" by another monoclonal antibody on silver nanoparticles labeled with fluorescein-labeled oligonucleotides in the presence of a magnet. Subsequently, the immune complex is enriched by exposure to a magnetic field. Lastly, the fluorescence intensity is measured according to the number of dissociated fluoresceins. The increased fluorescence intensity permits highly sensitive detection of SOD in a linear range of 10-8 × 10(5) pg mL(-1), with a detection limit of 4 pg mL(-1) at a signal-to-noise ratio of 3. Significantly, this method was validated for detection of SOD in human serum, human urine, and cosmetic samples. Moreover, the reliability and accuracy of results obtained by the enhanced-fluorescence method was confirmed by the analysis of high performance liquid chromatography (HPLC).

Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes

Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.

Genomic Selection Identifies Vertebrate Transcription Factor Fezf2 Binding Sites and Target Genes

Identification of transcription factor targets is critical to understanding gene regulatory networks. Here, we uncover transcription factor binding sites and target genes employing systematic evolution of ligands by exponential enrichment (SELEX). Instead of selecting randomly synthesized DNA oligonucleotides as in most SELEX studies, we utilized zebrafish genomic DNA to isolate fragments bound by Fezf2, an evolutionarily conserved gene critical for vertebrate forebrain development. This is, to our knowledge, the first time that SELEX is applied to a vertebrate genome. Computational analysis of bound genomic fragments predicted a core consensus binding site, which identified response elements that mediated Fezf2-dependent transcription both in vitro and in vivo. Fezf2-bound fragments were enriched for conserved sequences. Surprisingly, ∼20% of these fragments overlapped well annotated protein-coding exons. Through loss of function, gain of function, and chromatin immunoprecipitation, we further identified and validated eomesa/tbr2 and lhx2b as biologically relevant target genes of Fezf2. Mutations in eomesa/tbr2 cause microcephaly in humans, whereas lhx2b is a critical regulator of cell fate and axonal targeting in the developing forebrain. These results demonstrate the feasibility of employing genomic SELEX to identify vertebrate transcription factor binding sites and target genes and reveal Fezf2 as a transcription activator and a candidate for evaluation in human microcephaly.

Two Successive Reactions on a DNA Template: A Strategy for Improving Background Fluorescence and Specificity in Nucleic Acid Detection

We report a new strategy for template-mediated fluorogenic chemistry that results in enhanced performance for the fluorescence detection of nucleic acids. In this approach, two successive templated reactions are required to induce a fluorescence signal, rather than only one. These novel fluorescein-labeled oligonucleotide probes, termed 2-STAR (STAR = Staudinger-triggered α-azidoether release) probes, contain two quencher groups tethered by separate reductively cleavable linkers. When a 2-STAR quenched probe successively binds adjacent to two mono-triphenylphosphine-(TPP)-DNAs or one dual-TPP-DNA, the two quenchers are released, resulting in a fluorescence signal. Because of the requirement for two consecutive reactions, 2-STAR probes display an unprecedented level of sequence specificity for template-mediated probe designs. At the same time, background emission generated by off-template reactions or incomplete quenching is among the lowest of any fluorogenic reactive probes for the detection of DNA or RNA.

Simple surface modification of poly(dimethylsiloxane) for DNA hybridization.

Here, we present a simple chemical modification of poly(dimethylsiloxane) (PDMS) by curing a mixture of 2 wt% undecylenic acid (UDA) in PDMS prepolymer on a gold-coated glass slide. This gold slide had been previously pretreated with a self-assembled hydrophilic monolayer of 3-mercaptopropionic acid (MPA). During curing of the UDA∕PDMS prepolymer, the hydrophilic UDA carboxyl moieties diffuses toward the hydrophilic MPA carboxyl moieties on the gold surface. This diffusion of the UDA within the PDMS prepolymer to the surface is a direct result of surface energy minimization. Once completely cured, the PDMS is peeled off the gold substrate, thereby exposing the interfacial carboxyl groups. These groups are then available for subsequent attachment of 5(')-amino terminated DNA oligonucleotides via amide linkages. Our results show that the covalently tethered oligonucleotides can successfully capture fluorescein-labeled complementary oligonucleotides via hybridization, which are visualized using fluorescence microscopy.

Surpassing Specificity Limits of Nucleic Acid Probes via Cooperativity

The failure to correctly identify single nucleotide polymorphisms (SNPs) significantly contributes to the misdiagnosis of infectious disease. Contrary to the strategy of creating shorter probes to improve SNP differentiation, we created larger probes that appeared to increase selectivity. Specifically, probes with enhanced melting temperature differentials (>13x improvement) to SNPs were generated by linking two probes that consist of both a capture sequence and a detection sequence; these probes act cooperatively to improve selectivity over a wider range of reaction conditions. These cooperative probe constructs (Tentacle probes) were then compared by modeling thermodynamic and hybridization characteristics to both Molecular Beacons (stem loop DNA probes) and Taqman probes (a linear oligonucleotide). The biophysical models reveal that cooperative probes compared with either Molecular beacons or Taqman probes have enhanced specificity. This was a result of increased melting temperature differentials and the concentration-independent hybridization revealed between wild-type and variant sequences. We believe these findings of order of magnitude enhanced melting temperature differentials with probes possessing concentration independence and more favorable binding kinetics have the potential to significantly improve molecular diagnostic assay functionality.

Development and Evaluation of a Sensitive PCR-ELISA System for Detection of Schistosoma Infection in Feces

BackgroundA PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed to overcome the need for sensitive techniques for the efficient diagnosis of Schistosoma infection in endemic settings with low parasitic burden.Methodology/Principal FindingsThis system amplifies a 121-base pair tandem repeat DNA sequence, immobilizes the resultant 5′ biotinylated product on streptavidin-coated strip-well microplates and uses anti-fluorescein antibodies conjugated to horseradish peroxidase to detect the hybridized fluorescein-labeled oligonucleotide probe. The detection limit of the Schistosoma PCR-ELISA system was determined to be 1.3 fg of S. mansoni genomic DNA (less than the amount found in a single cell) and estimated to be 0.15 S. mansoni eggs per gram of feces (fractions of an egg). The system showed good precision and genus specificity since the DNA target was found in seven Schistosoma DNA samples: S. mansoni, S. haematobium, S. bovis, S. intercalatum, S. japonicum, S. magrebowiei and S. rhodaini. By evaluating 206 patients living in an endemic area in Brazil, the prevalence of S. mansoni infection was determined to be 18% by examining 12 Kato-Katz slides (41.7 mg/smear, 500 mg total) of a single fecal sample from each person, while the Schistosoma PCR-ELISA identified a 30% rate of infection using 500-mg of the same fecal sample. When considering the Kato-Katz method as the reference test, artificial sensitivity and specificity rates of the PCR-ELISA system were 97.4% and 85.1%, respectively. The potential for estimating parasitic load by DNA detection in feces was assessed by comparing absorbance values and eggs per gram of feces, with a Spearman correlation coefficient of 0.700 (P<0.0001).Conclusions/SignificanceThis study reports the development and field evaluation of a sensitive Schistosoma PCR-ELISA, a system that may serve as an alternative for diagnosing Schistosoma infection.

The DNA binding and 3′-end preferential activity of human tyrosyl-DNA phosphodiesterase

Human tyrosyl-DNA phosphodiesterase (Tdp1) processes 3′-blocking lesions, predominantly 3′-phosphotyrosyl bonds resulting from the trapping of topoisomerase I (Top1) cleavage complexes. The controversial ability of yeast Tdp1 to hydrolyze 5′-phosphotyrosyl linkage between topoisomerase II (Top2) and DNA raises the question whether human Tdp1 possesses 5′-end processing activity. Here we characterize the end-binding and cleavage preference of human Tdp1 using single-stranded 5′- and 3′-fluorescein-labeled oligonucleotides. We establish 3′-fluorescein as an efficient surrogate substrate for human Tdp1, provided it is attached to the DNA by a phosphodiester (but not a phosphorothioate) linkage. We demonstrate that human Tdp1 lacks the ability to hydrolyze a phosphodiester linked 5′-fluorescein. Using both fluorescence anisotropy and time-resolved fluorescence quenching techniques, we also show the preferential binding of human Tdp1 to the 3′-end. However, DNA binding competition experiments indicate that human Tdp1 binding is dependent on DNA length rather than number of DNA ends. Lastly, using surface plasmon resonance, we show that human Tdp1 selectively binds the 3′-end of DNA. Together, our results suggest human Tdp1 may act using a scanning mechanism, in which Tdp1 bind non-specifically upstream of a 3′-blocking lesion and is preferentially stabilized at 3′-DNA ends corresponding to its site of action.

Multiple Myeloma-Specific Targeted Therapy: Use of the Tumor Specific Immunoglobulin Gene Sequence to Activate Drugs Only within Tumor Cells.

Abstract 1851Poster Board I-877Currently available drugs for multiple myeloma (MM) treatment show very little ability to distinguish MM cells from normal cells. This limits the dose of drugs with anti-MM activity that can be administered safely; and, thus, reduces their efficacy in eliminating the malignant plasma cell population. Therefore, novel strategies are needed that allow concentrations of higher levels of active drugs within tumor cells than in other healthy cells. One approach is to allow release of active drugs only within the tumor cells in these patients without affecting nonmalignant cells. MM is a malignancy of clonal antibody-secreting plasma cells with specific rearrangement of DNA that is transcribed into a unique mRNA sequence that is translated into the tumor specific monoclonal antibody. These tumor specific transcripts are abundant in all of the malignant cells in these patients. We have explored the possibility of using a unique sequence from this tumor specific transcript, the complementarity determining region (CDR) gene sequence, to direct drug release only within MM cells. First, we determined whether this type of tumor specific oligonucleotide could specifically recognize the tumor cell population. Using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from the MM cell line RPMI8226 as a probe (designated as molecular beacon [MB] 8226), we demonstrated that this oligonucleotide specifically distinguished the RPMI8226 from other cell lines, including U266, another MM cell line. Second, in order to translate this approach into potentially therapeutically active anti-MM agents, we synthesized naphthyridine-modified (N) vorinostat, a FDA-approved histone deacetylase inhibitor, and N-melphalan. The modification allowed us to conjugate vorinostat or melphalan, two drugs currently used in MM treatment, with MB8226. Histone acetylation analysis demonstrated that the modification of vorinostat with naphthyridine did not change its function compared to the unmodified compound. Moreover, using cell viability and apoptosis assays, there was no reduction in the cytotoxic effects of N-vorinostat or N-melphalan compared to the parent drugs. In addition, induction of cell death occurred in a cell specific matter with N-melphalan conjugated to its tumor specific oligonucleotide. Upon transfection with N-melphalan-conjugated MB8226, only RPMI8226 but not U266 cells showed cytotoxic effects from exposure to this tumor specific cytotoxic construct. We are currently evaluating the specific efficacy of N-vorinostat-conjugated MB8226 as well as an oligonucleotide that specifically recognizes the CDR3 sequence of U266 cells conjugated with N-vorinostat or N-melphalan. We are also determining the efficacy of this approach in vivo using our severe combined immunodeficiency-hu mouse models of human MM. Our studies provide a novel targeted therapeutic approach for the treatment MM as well as other B cell malignancies that should specifically be active only within the malignant cell population and not impact nonmalignant cells. This type of treatment should allow delivery of higher concentrations of drugs that will be active only within tumor cells ultimately leading to both much more effective and better tolerated therapies for patients with MM and other B-cell malignancies. Disclosures:No relevant conflicts of interest to declare.

Synthesis and Studies on Cell-Penetrating Peptides

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.

Role of Bound Zn(II) in the CadC Cd(II)/Pb(II)/Zn(II)-responsive Repressor

The Staphylococcus aureus plasmid pI258 cadCA operon encodes a P-type ATPase, CadA, that confers resistance to Cd(II)/Pb(II)/Zn(II). Expression is regulated by CadC, a homodimeric repressor that dissociates from the cad operator/promoter upon binding of Cd(II), Pb(II), or Zn(II). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins. The crystal structure of CadC shows two types of metal binding sites, termed Site 1 and Site 2, and the homodimer has two of each. Site 1 is the physiological inducer binding site. The two Site 2 metal binding sites are formed at the dimerization interface. Site 2 is not regulatory in CadC but is regulatory in the homologue SmtB. Here the role of each site was investigated by mutagenesis. Both sites bind either Cd(II) or Zn(II). However, Site 1 has higher affinity for Cd(II) over Zn(II), and Site 2 prefers Zn(II) over Cd(II). Site 2 is not required for either derepression or dimerization. The crystal structure of the wild type with bound Zn(II) and of a mutant lacking Site 2 was compared with the SmtB structure with and without bound Zn(II). We propose that an arginine residue allows for Zn(II) regulation in SmtB and, conversely, a glycine results in a lack of regulation by Zn(II) in CadC. We propose that a glycine residue was ancestral whether the repressor binds Zn(II) at a Site 2 like CadC or has no Site 2 like the paralogous ArsR and implies that acquisition of regulatory ability in SmtB was a more recent evolutionary event.

Quantum dots thermal stability improves simultaneous phenotype-specific telomere length measurement by FISH-flow cytometry

Telomere length analysis has been greatly simplified by the quantitative flow cytometry technique FISH-flow. In this method, a fluorescein-labeled synthetic oligonucleotide complementary to the telomere terminal repeat sequence is hybridized to the telomere sequence and the resulting fluorescence measured by flow cytometry. This technique has supplanted the traditional laborious Southern blot telomere length measurement techniques in many laboratories, and allows single cell analysis of telomere length in high-throughput sample formats. Nevertheless, the harsh conditions required for telomere probe annealing (82 °C) has made it difficult to successfully combine this technique with simultaneous immunolabeling. Most traditional organic fluorescent probes (i.e. fluorescein, phycoerythrin, etc.) have limited thermal stability and do not survive the high temperature annealing process, despite efforts to covalently crosslink the antigen–antibody–fluorophore complex. This loss of probe fluorescence has made it difficult to measure FISH-flow in complex lymphocyte populations, and has generally forced investigators to use fluorescent-activated cell sorting to pre-separate their populations, a laborious technique that requires prohibitively large numbers of cells.In this study, we have substituted quantum dots (nanoparticles) for traditional fluorophores in FISH-flow. Quantum dots were demonstrated to possess much greater thermal stability than traditional low molecular weight and phycobiliprotein fluorophores. Quantum dot antibody conjugates directed against monocyte and T cell antigens were found to retain most of their fluorescence following the high temperature annealing step, allowing simultaneous fluorescent immunophenotyping and telomere length measurement. Since quantum dots have very narrow emission bandwidths, we were able to analyze multiple quantum dot antibody conjugates (Qdot 605, 655 and 705) simultaneously with FISH-flow measurement to assess the age-associated decline in telomere length in both human monocytes and T cell subsets. With quantum dot immunolabeling, the mean decrease rate in telomere length for CD4+ cells was calculated at 41.8 bp/year, very close to previously reported values using traditional flow-FISH and Southern blotting. This modification to the traditional flow-FISH technique should therefore allow simultaneous fluorescent immunophenotyping and telomere length measurement, permitting complex cell subset-specific analysis in small numbers of cells without the requirement for prior cell sorting.

Synthesis and Cellular Uptake of Fluorescently Labeled Multivalent Hyaluronan Disaccharide Conjugates of Oligonucleotide Phosphorothioates

Clustered hyaluronan disaccharides were studied as mediators of cellular delivery of antisense oligonucleotides through receptor-mediated endocytosis. For this purpose, a synthetic route for preparation of an appropriately protected hyaluronic acid dimer bearing an aldehyde tether (1) was devised. Up to three non-nucleosidic phosphoramidite building blocks (2), each bearing two phthaloyl protected aminooxy groups, were then inserted into the 3'-terminus of the desired phosphorothioate oligodeoxyribonucleotide, and 6-FAM phosphoramidite was introduced into the 5'-terminus. After completion of the chain assembly, the aldehyde-tethered sugar ligands were attached to the deprotected aminooxy functions by on-support oximation. Three fluorescein-labeled phosphorothioate oligonucleotide glycoconjugates (28-30) containing two, four, or six hyaluronan disaccharides were prepared. The influence of the hyaluronan moieties on the cellular uptake of the thioated oligonucleotides was tested in a cell line expressing the hyaluronan receptor CD44. Specific uptake was not detected with this combination of multiple hyaluronan disaccharides.

Simultaneous Phenotype-Specific Telomere Length Measured by Flow-FISH Using Thermally Stable Quantum Dots

The Flow-FISH technique used to measure telomere length by incorporation of fluorescein-labeled oligonucleotide probes complementary to telomeres repeat sequences has been extended to permit simultaneous multiple phenotypic analysis within a single sample. The necessary thermal stability of the fluorescent labeling needed for phenotyping cell populations can be achieved using quantum dots instead of conventional organic fluorophores, which are usually damaged during the high-temperature hybridization treatment and lose their fluorescence, limiting their usefulness for phenotypic analysis. In this study, quantum dots and conventional fluorophores were compared for their ability to survive the 82oC hybridization step necessary for PNA probe annealing. Quantum dots preserved their fluorescence following heat treatment and gave good signal-to-noise ratios when used to detect lymphocyte and monocyte markers (CD3, CD4 and CD14). This was in contrast to phycobiliproteins and low molecular weight fluorochromes such as fluorescein and the Alexa Fluor dyes, which decreased considerably in fluorescence following hybridization. Since addition of fluorescent immunophenotyping complicated an assay already heavily dependent on uniform technique, internal reference standards (previously reported by Lansdorp) were particularly critical in this technique. We used aliquoted frozen calf thymocytes (CT) as an internal standard, determining their telomere length and incorporating them into each sample to monitor tube to tube and day to day assay variability. The telomere length of the CTs and of CD3+CD4+, CD3+CD4− and CD14+, were determined and then corrected by normalization to the known CT telomere length. To validate the overall method, we then measured age dependence on telomere length for monocyte and lymphocyte subpopulations in young, middle-aged and older donors. Results were consistent with results previously reported using traditional Southern blotting and Flow-FISH with no immunphenotyping, suggesting that incorporation of immunolabeling did not adversely affect this technique. This method allows for the rapid, accurate and simultaneous determination of the telomere lengths of different cell populations within a single sample when little amount of sample is available.

A Novel Targeted Therapeutic Approach to Multiple Myeloma Using Tumor Specific Anti-Sense RNA Sequences

Despite recent advances in the therapy of multiple myeloma (MM), the disease remains incurable. Current drugs for MM target not only MM but also normal cells. This limits the efficacy of these agents and may lead to significant morbidities for patients. Therefore, novel strategies are needed that allow targeting of tumor cells in these patients without affecting nonmalignant cells. MM is a malignancy of clonal antibody-secreting plasma cells with specific rearrangement of DNA that is transcribed into a unique mRNA sequence that is translated into the tumor specific monoclonal antibody. These tumor specific transcripts are abundant in all of the malignant cells of these patients. We have explored the possibility of using a unique sequence from this transcript, the complementarity determining region (CDR) gene sequence, to direct drug release into MM cells. Using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from the MM cell line RPMI8226 as a probe, our results have demonstrated that quenched fluorescent signals of this RPMI8226-specific oligonucleotide were released upon binding to the CDR3 gene sequence of RPMI8226, but not to oligo(dT), random sequences or the CDR3 gene sequence from another MM cell line, U266. Similarly, using a quenched fluorescein-labeled oligonucleotide sequence complementary to the CDR3 gene sequence from U266 as a probe, our results have also shown that quenched fluorescent signals of the U266-specific oligonucleotide were released upon binding to the CDR3 gene sequence of U266, but not to oligo(dT), random sequences or the CDR3 gene sequence from RPMI8226. In addition, transfection of 8226- or U266-specific oligonucleotides into 8226 and U266 cells indicated that the oligonucleotides specifically discriminated the two cell lines. Furthermore, these oligonucleotides are stable in PBS or in transfected cells for at least 48hrs at 37°C, long enough for killing of MM cells with most currently available anti-MM drugs. Currently, we are evaluating the efficacy of this targeted approach to specifically identify tumor cells, and developing therapeutic modification of the oligonucleotide to allow drug delivery specifically to the tumor cells. Our studies validate a new strategy for MM treatment that may introduce a new targeted therapeutic approach in MM as well as other B cell malignancy, without impacting nonmalignant cells, which should allow much higher concentrations of drugs to be delivered into the tumor cells. Updated results using this novel targeted treatment approach will be reported at the meeting.

The TREX1 Double-stranded DNA Degradation Activity Is Defective in Dominant Mutations Associated with Autoimmune Disease

Mutations in TREX1 have been linked to a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome (AGS), familial chilblain lupus (FCL), systemic lupus erythematosus, and retinal vasculopathy and cerebral leukodystrophy. A common feature in these conditions is the frequent detection of antibodies to double-stranded DNA (dsDNA). TREX1 participates in a cell death process implicating this major 3' --> 5' exonuclease in genomic DNA degradation to minimize potential immune activation by persistent self DNA. The TREX1 D200N and D18N dominant heterozygous mutations were identified in AGS and FCL, respectively. TREX1 enzymes containing the D200N and D18N mutations were compared using nicked dsDNA and single-stranded DNA (ssDNA) degradation assays. The TREX1WT/D200N and TREX1WT/D18N heterodimers are completely deficient at degrading dsDNA and degrade ssDNA at an expected approximately 2-fold lower rate than TREX1WT enzyme. Further, the D200N- and D18N-containing TREX1 homo- and heterodimers inhibit the dsDNA degradation activity of TREX1WT enzyme, providing a likely explanation for the dominant phenotype of these TREX1 mutant alleles in AGS and FCL. By comparison, the TREX1 R114H homozygous mutation causes AGS and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1R114H/R114H homodimer has dysfunctional dsDNA and ssDNA degradation activities and does not detectibly inhibit the TREX1WT enzyme, whereas the TREX1WT/R114H heterodimer has a functional dsDNA degradation activity, supporting the recessive genetics of TREX1 R114H in AGS. The dysfunctional dsDNA degradation activities of these disease-related TREX1 mutants could account for persistent dsDNA from dying cells leading to an aberrant immune response in these clinically related disorders.