Bioluminescent reporters for identification of gene allelic variants

A method for single nucleotide polymorphism identification was developed, which was based on the primer extension reaction (PEXT) followed by bioluminescent solid-phase microassay. Recombinant Ca²⁺-regulated photoprotein obelin and coelenterazine-dependent Renilla muelleri luciferase were used as reporters. The study was performed as an example of SNP genotyping of the human F5 gene encoding human Factor V Leiden polymorphism 1691 G??A (R506Q). Genomic DNA was amplified by PCR using primers flanking polymorphic site of 140 base pairs. PCR products were used as templates for two PEXT reactions using two primers containing 3??-terminal nucleotides, which were complementary to either normal or mutant alleles. If the template and allele-specific primer were completely complementary, the latter was elongated with DNA polymerase. The resulting extension product contained biotin residue due to the presence of biotinylated deoxyuridine triphosphate (B-dUTP) in the reaction mixture. The products were analyzed using obelin-streptavidin conjugates. The optimal PEXT-reaction conditions were found, which ensured a high reliability of SNP genotyping. A new approach to simultaneously revealing both alleles in one well was developed using two bioluminescent reporters. The efficiency of the proposed approach was shown in the study of clinical DNA samples.     

The Ca<Superscript>2+</Superscript>-Regulated Photoprotein Obelin as a Target for the RNA Aptamer Selection

A variant of the Ca²⁺-regulated photoprotein obelin elongated with a hexahistidine peptide from the N-terminus was developed and studied. After immobilization on a metal-affine sorbent, the hybrid protein was applied as a target for the in vitro selection of RNA aptamers. According to the data of bioluminescent solid-phase microanalysis, the selection was shown to enrich the RNA library with obelin-affine molecules.     

Development of a colorimetric test system for detection of point mutations via ligation of a tandem of short oligonucleotides on methacrylate beads

A new approach for detection of point mutations has been developed. The nonradioactive test system proposed is based on enzymatic ligation of a tandem of three short oligonucleotides B∼pN₈+pN₄+pN′₈ Bio in the presence of a complementary DNA template. The 5′-terminal octanucleotide B∼pN₈ is immobilized on polymer methacrylate beads (B) and the 3′-terminal octanucleotide pN′₈ Bio contains a biotin residue at the 3′-phosphate. Ligation of the tandem produces a 20-mer biotinylated oligonucleotide on a polymer bead, which is then visualized via subsequent treatments with streptavidin-alkaline phosphatase conjugate and chromogenic substrates. Intense staining of the polymer beads is observed when the amount of DNA template (20-mer oligonucleotide) is as low as ∼10⁻¹⁴ mol. It is shown that practically no polymer staining is observed when the complex formed by the tandem and the 20-mer DNA template contains a mismatch either in the tetranucleotide duplex or in the duplex of octanucleotide immobilized on the beads. This suggests a possibility of using the presented approach in test systems for detection of point mutations in PCR-amplified DNA fragments.     

Single-Stranded DNA-Binding Protein Complex from Helicobacter pylori Suggests an ssDNA-Binding Surface

Single-stranded DNA (ssDNA)-binding protein (SSB) plays an important role in DNA replication, recombination, and repair. SSB consists of an N-terminal ssDNA-binding domain with an oligonucleotide/oligosaccharide binding fold and a flexible C-terminal tail involved in protein-protein interactions. SSB from Helicobacter pylori (HpSSB) was isolated, and the ssDNA-binding characteristics of HpSSB were analyzed by fluorescence titration and electrophoretic mobility shift assay. Tryptophan fluorescence quenching was measured as 61%, and the calculated cooperative affinity was 5.4 × 10⁷ M^− 1 with an ssDNA-binding length of 25-30 nt. The crystal structure of the C-terminally truncated protein (HpSSBc) in complex with 35-mer ssDNA [HpSSBc-(dT)₃₅] was determined at a resolution of 2.3 Å. The HpSSBc monomer folds as an oligonucleotide/oligosaccharide binding fold with a Y-shaped conformation. The ssDNA wrapped around the HpSSBc tetramer through a continuous binding path comprising five essential aromatic residues and a positively charged surface formed by numerous basic residues.     

Development of non-electrophoretic assay method for DNA ligases and its application to screening of chemical inhibitors of DNA ligase I

A new rapid assay method for DNA ligases has been developed, which allows direct quantification of enzyme activity without using the traditional polyacrylamide gel electrophoretic technique. In this method, the 5'-biotinylated nicked duplex was used as a substrate for the ligase reaction, in which the 5'-end of the first oligonucleotide (19-mer) on the nicked strand is biotinylated and the second oligonucleotide (20-mer) on the same strand is labeled with radioactive 32P at the 5'-end. After ligation of the biotinylated 19-mer oligonucleotide into the second oligonucleotide with the reaction of DNA ligases, the biotinylated 19-mer oligonucleotide is converted into the radioactive 39-mer oligonucleotide. The ligase reaction products were heat-denatured to release both ligated and unligated biotinylated oligonucleotides. The biotinylated oligonucleotides were then captured on a streptavidin-coated microtiter plate and counted. The results obtained using this method correlated very well with those from the standard assay method using electrophoresis. Using this assay method, we were able to screen a chemical library and identify new DNA ligase inhibitors structurally related to resorcinol, which has growth inhibitory effects on the human breast cancer cell, MCF-7. The method described here is anticipated to be very useful for screening DNA ligase inhibitors from chemical libraries.     

A New Approach to Detect a Particular DNA Sequence by UV-Immobilization of Its Hybridization Complex with a Highly Specific Probe Resulting from Ligation of a Tandem of Short Oligonucleotides in Solution

A new approach was proposed for detecting amplified DNA fragments by hybridization with a highly selective oligonucleotide probe obtained by ligation of a tandem of three short oligonucleotides (pN₈ + pN₄ + pN₈ ^"(Bio)) in solution, with subsequent UV-immobilization of the hybridization product on a nylon membrane and its colorimetric detection with the streptavidin–alkaline phosphatase technique. Owing to the high selectivity of ligation, the 20-mer ligation product was detected on a membrane only when it was fully complementary to a template fragment. The results showed that any single-nucleotide substitution in the tetramer-binding site can be localized and identified with the use of all 12 possible tetramers.     

Genetically engineered obelin as a bioluminescent label in an assay for a peptide.

The marine polyp Obelia longissima produces a protein, obelin, which emits light in a calcium-dependent manner. This photoprotein consists of a stable complex of its apoprotein, a chromophore, and oxygen. In the presence of calcium ions, the protein undergoes a change in conformation that allows it to catalyze the oxidation of the chromophore, coelenterazine, to coelenteramide with the release of light and CO2. Photoproteins are attractive as labels in analytical applications because the bioluminescent signal that they produce is the result of a chemical reaction and, therefore, has virtually no background. Thus, bioluminescence allows for extremely sensitive detection. In that regard, the feasibility of using obelin as a label has been explored with the development of a competitive immunoassay for the determination of a small peptide analyte. To attach the obelin label in a controlled manner to the octapeptide, a fusion protein was produced using recombinant DNA techniques. The protein consisted of the C-terminus of the peptide fused to the N-terminus of obelin. The octapeptide-obelin fusion protein retained the bioluminescence properties of the native protein, and was subsequently used to generate dose-response curves for the free octapeptide.     

Conjugates of the Сa2+-Regulated Photoprotein Obelin with Immunoglobulins: Synthesis and Use as Labels in Bioluminescent Immunoassay

An efficient procedure for obelin conjugation with immunoglobulins was developed. The possibility was shown of using the resulting conjugates instead of a radioisotope label for the immunoassay of thyroid stimulating hormone in sera; the conjugates provide a sensitivity of 0.01 IU/ml. The results of bioluminescent immunoassay (sera of 34 patients) satisfactorily correlate with the results of radioisotope assay (R 0.99).     

Apoptotic endonuclease EndoG regulates alternative splicing of human telomerase catalytic subunit hTERT

Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of β-deletion splice variant was determined during EndoG overexpression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei as well as after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.     

Barnase-barstar high affinity interaction phenomenon as the base for the heterogenous bioluminescence pseudorabies virus' immunoassay

The effective new variant of "sandwich" bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase-barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-"sandwich" principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.     

Production of random DNA oligomers for scalable DNA computing

While remarkably complex networks of connected DNA molecules can form from a relatively small number of distinct oligomer strands, a large computational space created by DNA reactions would ultimately require the use of many distinct DNA strands. The automatic synthesis of this many distinct strands is economically prohibitive. We present here a new approach to producing distinct DNA oligomers based on the polymerase chain reaction (PCR) amplification of a few random template sequences. As an example, we designed a DNA template sequence consisting of a 50-mer random DNA segment flanked by two 20-mer invariant primer sequences. Amplification of a dilute sample containing about 30 different template molecules allows us to obtain around 10¹¹ copies of these molecules and their complements. We demonstrate the use of these amplicons to implement some of the vector operations that will be required in a DNA implementation of an analog neural network.     

Conjugates of the Ca2+ -regulated photoprotein obelin with immunoglobulins: synthesis and use as labels in bioluminescent immunoassay

An efficient procedure for obelin conjugation with immunoglobulins was developed. The possibility was shown of using the resulting conjugates instead of a radioisotope label for the immunoassay of thyroid stimulating hormone in sera; the conjugates provide a sensitivity of 0.01 microIU/ml. The results of bioluminescent immunoassay (sera of 34 patients) satisfactorily correlate with the results of radioisotope assay (R 0.99).     

Barnase–barstar high affinity interaction phenomenon as the base for the heterogenous bioluminescence pseudorabies virus' immunoassay

The effective new variant of “sandwich” bioluminescent enzyme immunoassay (BEIA) for the sensitive detection of glycoprotein B (gB) of pseudorabies virus (PrV) was presently developed. The high affinity interaction of barnase–barstar protein pair and photoprotein obelin as bioluminescent marker were for the first time successfully applied to BEIA development. Preliminary the two monoclonal antibodies, 11/5 and 34/2, were raised against gB for ELISA PrV detection. Presently we used the same immuno-“sandwich” principle for BEIA. To do this the two different bioconjugates were elaborated. Recombinant barnase was chemically conjugated with monoclonal anti-PrV's gB IgG, and also barstar was fused in frame to obelin. The characteristics of BEIA method have been compared to ELISA PrV detection. We have shown the proposed here gB-BEIA was 40-fold more sensitive as opposed to gB-ELISA test. The construction might have a broad promise in multiple potential immunological applications.     

Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance

A DNA piezoelectric biosensing method for real-time detection of Escherichia coli O157:H7 in a circulating-flow system was developed in this study. Specific probes [a 30-mer oligonucleotide with or without additional 12 deoxythymidine 5′-monophosphate (12-dT)] for the detection of E. coli O157:H7 gene eaeA, synthetic oligonucleotide targets (30 and 104 mer) and PCR-amplified DNA fragments from the E. coli O157:H7 eaeA gene (104 bp), were used to evaluate the efficiency of the probe immobilization and hybridization with target DNA in the circulating-flow quartz crystal microbalance (QCM) device. It was found that thiol modification on the 5′-end of the probes was essential for probe immobilization on the gold surface of the QCM device. The addition of 12-dT to the probes as a spacer, significantly enhanced (P < 0.05) the hybridization efficiency (H%). The results indicate that the spacer enhanced the H% by 1.4- and 2-fold when the probes were hybridized with 30- and 104-mer targets, respectively. The spacer reduced steric interference of the support on the hybridization behavior of immobilized oligonucleotides, especially when the probes hybridized with relatively long oligonucleotide targets. The QCM system was also applied in the detection of PCR-amplified DNA from real samples of E. coli O157:H7. The resultant H% of the PCR-amplified double-strand DNA was comparable to that of the synthetic target T-104AS, a single-strand DNA. The piezoelectric biosensing system has potential for further applications. This approach lays the groundwork for incorporating the method into an integrated system for rapid PCR-based DNA analysis.     

Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

Background

The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR.

Results

A liposome detection reagent was prepared, which consisted of a population of liposomes ~120?nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose?Cresponse curve from 10^-10?M to 10^-16?M CEA. Within this range the assay coefficient of variance was <6?% for repeatability and <2?% for reproducibility. The assay detection limit was 13?fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed.

Conclusions

The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to encapsulate multiple reporters per liposome also helps overcome the effect of polymerase inhibitors present in biological specimens. Finally, the biotin-labeled liposome detection reagent can be coupled through a NeutrAvidin bridge to a multitude of biotin-labeled probes, making ILPCR a highly generic assay system.     

Quantitative measurement of cell membrane receptor internalization by the nanoluciferase reporter: Using the G protein-coupled receptor RXFP3 as a model

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence to date. In the present work, we developed NanoLuc as a sensitive bioluminescent reporter to measure quantitatively the internalization of cell membrane receptors, based on the pH dependence of the reporter activity. The G protein-coupled receptor RXFP3, the cognate receptor of relaxin-3/INSL7, was used as a model receptor. We first generated stable HEK293T cells that inducibly coexpressed a C-terminally NanoLuc-tagged human RXFP3 and a C-terminally enhanced green fluorescent protein (EGFP)-tagged human RXFP3. The C-terminal EGFP-tag and NanoLuc-tag had no detrimental effects on the ligand-binding potency and intracellular trafficking of RXFP3. Based on the fluorescence of the tagged EGFP reporter, the ligand-induced RXFP3 internalization was visualized directly under a fluorescence microscope. Based on the bioluminescence of the tagged NanoLuc reporter, the ligand-induced RXFP3 internalization was measured quantitatively by a convenient bioluminescent assay. Coexpression of an EGFP-tagged inactive [E141R]RXFP3 had no detrimental effect on the ligand-binding potency and ligand-induced internalization of the NanoLuc-tagged wild-type RXFP3, suggesting that the mutant RXFP3 and wild-type RXFP3 worked independently. The present bioluminescent internalization assay could be extended to other G protein-coupled receptors and other cell membrane receptors to study ligand –receptor and receptor–receptor interactions.     

Destabilization of Zn coordination in ADP-ribose transferase (polymerizing) by 6-nitroso-1,2-benzopyrone coincidental with inactivation of the polymerase but not the DNA binding function

6-Nitroso- 1,2-benzopyrone, an oxidation product of 6-amino- 1,2-benzopyrone, binds to the DNA-recognizing domain of the ADP-ribose transferase protein and preferentially destabilizes Zn²⁺ from one of the two zinc finger polypeptide complexes present in the intact enzyme, as determined by the loss of 50% of ⁶⁵Zn²⁺ from the ⁶⁵Zn²⁺-isolated protein molecule, coincidental with the loss of 99% of enzymatic activity. The 50% zinc-deficient enzyme still binds to a DNA template. consisting of a 17-mer DNA primer annealed to M 13 positive strand, resulting in the blocking of DNA synthesis by the Klenow fragment of Pol I, Auto-poly-ADP-ribosylated ADP-ribose transferase, which is the probable physiological state of this protein in intact cells, does not bind to primer-template DNA and does not block DNA synthesis by the Klenow fragment. On the basis of this in vitro model it is proposed that molecules which inhibit or inactivate ADP-ribose transferase in intact cells can induce significant alteration in DNA structure and replication.