Design of Coelenterazine Analogue to Reveal Bioluminescent Reaction of Human Serum Albumin

This chapter describes the design of an imidazopyrazinone-type luciferin named as HuLumino1 by us and investigation of its luminescence properties. This luciferin was designed to generate bioluminescence by human serum albumin (HSA) rather than by luciferase derived from luminous organisms. HuLumino1 was developed by modifying a methoxy-terminated alkyl chain to the C-6 position and eliminating a benzyl group at the C-8 position of coelenterazine. To clarify the basis of light emission by HSA, the detailed kinetic properties of the HuLumino1/HSA pair were investigated using a calibrated luminometer. The enzymatic oxidation of HuLumino1 was observed only in the presence of HSA. Results of HSA quantification experiments using HuLumino1 agreed with less than 5% differences with those of enzyme-linked immunosorbent assays, suggesting HuLumino1 could be used for quantitative analysis of HSA levels in serum samples without any pretreatments. These results demonstrate the advantages of the coelenterazine analogue as a bioluminescence reagent to detect non-labeled proteins, which generally do not function as enzymes

A polymer-based chemical tongue for the non-invasive monitoring of osteogenic stem-cell differentiation by pattern recognition of serum-supplemented spent media

The development of non-invasive techniques to characterize cultured cells is invaluable not only to ensure the reproducibility of cell research, but also for quality assurance of industrial cell products for purposes such as regenerative medicine. Here, we present a polymer-based ‘chemical tongue’, i.e., a biosensing technique that mimics the human taste system, that is capable of non-invasively generating fluorescence response patterns that reflect the proteins secreted, and also partially consumed, by cultured cells, even from serum-supplemented media containing abundant interferants. Analysis of the spent media collected during cell culture using our chemical tongue, which consists of cationic polymers of different scaffolds appended with environmentally responsive dansyl fluorophores, led to the successful (i) identification of human-derived cell lines, (ii) monitoring of the differentiation process of stem cells, even at the stage when conventional staining was negative, and (iii) detection of cancer-cell contamination in stem cells. Since the characterization of cultured cells is usually performed via invasive methods that result in cell death, our chemical-tongue approach, which is of high practical utility, will offer a new means of addressing the growing demand for highly controlled cell production in the medical and environmental fields

A Multi-Fluorescent DNA/Graphene Oxide Conjugate Sensor for Signature-Based Protein Discrimination

Signature-based protein sensing has recently emerged as a promising prospective alternative to conventional lock-and-key methods. However, most of the current examples require the measurement of optical signals from spatially-separated materials for the generation of signatures. Herein, we present a new approach for the construction of multi-fluorescent sensing systems with high accessibility and tunability, which allows generating protein fluorescent signatures from a single microplate well. This approach is based on conjugates between nano-graphene oxide (nGO) and three single-stranded DNAs (ssDNAs) that exhibit different sequences and fluorophores. Initially, the three fluorophore-modified ssDNAs were quenched simultaneously by binding to nGO. Subsequent addition of analyte proteins caused a partial recovery in fluorescent intensity of the individual ssDNAs. Based on this scheme, we have succeeded in acquiring fluorescence signatures unique to (i) ten proteins that differ with respect to pI and molecular weight and (ii) biochemical marker proteins in the presence of interferent human serum. Pattern-recognition methods demonstrated high levels of discrimination for this system. The high discriminatory power and simple format of this sensor system should enable an easy and fast evaluation of proteins and protein mixtures

DNA Methylation Analysis Triggered by Bulge Specific Immuno-Recognition

We report the sequence-selective discrimination of the cytosine methylation status in DNA with anti methylcytosine antibody for the first time. This was realized by employing an affinity measurement involving the target methylcytosine in a bulge region and anti methylcytosine antibody, following hybridization with a bulge-inducing DNA to ensure that only the target methylcytosine is located in the bulge. The affinity of the antibody for methylcytosine in the bulge was 79% of that in a single strand of DNA; however, the affinity for nontarget methylcytosine in a double strand of DNA decreased greatly. This is because the antibody cannot bind with an inwardly turned methylcytosine in the duplex region owing to the large antibody size. In contrast, the methylcytosine in the bulge is recognized by the antibody because it is available to rotate freely owing to the single bond between deoxyribose and phosphate in a DNA chain. By employing the difference between the affinity in the bulge and that in the duplex, we could determine selectively whether or not the target cytosine was methylated in an O⁶-methylguanine DNA methyltransferase (MGMT) promoter sequence with a single base level. The proposed bulge-specific assay technique can be combined with a widely used absorbance measurement method that employs the color change in tetramethyl benzidine induced by horseradish peroxidase-labeled secondary antibody. The sequence-selective discrimination of the methylation status could also be obtained with various types of interfering genomic DNA contamination without any conventional bisulfite treatment, polymerase chain reaction, (PCR) or electrophoresis

N6-Methylation Assessment in <i>Escherichia coli</i> 23S rRNA Utilizing a Bulge Loop in an RNA–DNA Hybrid

We propose a sequence-selective assay of N6-methyl-adenosine (m6A) in RNA without PCR or reverse transcription, by employing a hybridization assay with a DNA probe designed to form a bulge loop at the position of a target modified nucleotide. The m6A in the bulge in the RNA–DNA hybrid was assumed to be sufficiently mobile to be selectively recognized by an anti-m6A antibody with a high affinity. By employing a surface-plasmon-resonance measurement or using a microtiter-plate immunoassay method, a specific m6A in the <i>Escherichia coli</i> 23S rRNA sequence could be detected at the nanomolar level when synthesized and purified oligo-RNA fragments were used for measurement. We have successfully achieved the first selective detection of m6A₂₀₃₀ specifically in 23S rRNA from real samples of <i>E. coli</i> total RNA by using our immunochemical approach

A Multichannel Pattern-Recognition-Based Protein Sensor with a Fluorophore-Conjugated Single-Stranded DNA Set

Recently, pattern-recognition-based protein sensing has received considerable attention because it offers unique opportunities that complement more conventional antibody-based detection methods. Here, we report a multichannel pattern-recognition-based sensor using a set of fluorophore-conjugated single-stranded DNAs (ssDNAs), which can detect various proteins. Three different fluorophore-conjugated ssDNAs were placed into a single microplate well together with a target protein, and the generated optical response pattern that corresponds to each environment-sensitive fluorophore was read via multiple detection channels. Multivariate analysis of the resulting optical response patterns allowed an accurate detection of eight different proteases, indicating that fluorescence signal acquisition from a single compartment containing a mixture of ssDNAs is an effective strategy for the characterization of the target proteins. Additionally, the sensor could identify proteins, which are potential targets for disease diagnosis, in a protease and inhibitor mixture of different composition ratios. As our sensor benefits from simple construction and measurement procedures, and uses accessible materials, it offers a rapid and simple platform for the detection of proteins

Epigenetic regulation of the circadian clock: Role of 5-aza-2&apos;-deoxycytidine

ABSTRACT We have been investigating transcriptional regulation of the BMAL1 gene, a critical component of the mammalian clock system including DNA methylation. Here, a more detailed analysis of the regulation of DNA methylation of BMAL1 proceeded in RPMI8402 lymphoma cells. We found that CpG islands in the BMAL1 and the PER2 promoters were hyper-and hypo-methylated, respectively and that 5-aza-2&apos;-deoxycytidine (aza-dC) not only enhanced PER2 gene expression but also PER2 oscillation within 24 hours in RPMI8402 cells. That is, such hypermethylation of CpG islands in the BMAL1 promoter restricted PER2 expression that was recovered by aza-dC within one day in these cells. These results suggest that the circadian clock system can be recovered through BMAL1 expression induced by aza-dC within a day. The RPIB9 promoter of RPMI8402 cells, which is a methylation hotspot in lymphoblastic leukemia, was also hypermethylated, and aza-dC gradually recovered RPIB9 expression in three days. In addition, methylation-specific PCR revealed a different degree of aza-dC-induced methylation release by between BMAL1 and RPIB9. These results suggest that the aza-dC-induced recovery of gene expression from DNA methylation is dependent on a gene, for example, the rapid response to demethylation by the circadian system, and thus is of importance to clinical strategies for treating cancer