ABSTRACT
The rapid development of messenger RNA (mRNA) vaccines formulated with lipid nanoparticles (LNPs) has contributed to control of the COVID-19 pandemic. However, mRNA vaccines have raised concerns about their potential toxicity and clinical safety, including side effects, such as myocarditis, anaphylaxis, and pericarditis. In this study, we investigated the potential of trehalose glycolipids-containing LNP (LNP S050L) to reduce the risks associated with ionizable lipids. Trehalose glycolipids can form hydrogen bonds with polar biomolecules, allowing the formation of a stable LNP structure by replacing half of the ionizable lipids. The efficacy and safety of LNP S050L were evaluated by encapsulating the mRNA encoding the luciferase reporter gene and measuring gene expression and organ toxicity, respectively. Furthermore, mice immunized with an LNP S050L-formulated mRNA vaccine expressing influenza hemagglutinin exhibited a significant reduction in organ toxicity, including in the heart, spleen, and liver, while sustaining gene expression and immune efficiency, compared to conventional LNPs (Con-LNPs). Our findings suggest that LNP S050L, a trehalose glycolipid-based LNP, could facilitate the development of safe mRNA vaccines with improved clinical safety.
ABSTRACT
The implications of administration of mRNA vaccines to individuals with chronic inflammatory diseases, including myocarditis, rheumatoid arthritis (RA), and inflammatory bowel disease (IBD), are unclear. We investigated mRNA vaccine effects in a chronic inflammation mouse model implanted with an LPS pump, focusing on toxicity and immunogenicity. Under chronic inflammation, mRNA vaccines exacerbated cardiac damage and myocarditis, inducing mild heart inflammation with heightened pro-inflammatory cytokine production and inflammatory cell infiltration in the heart. Concurrently, significant muscle damage occurred, with disturbances in mitochondrial fusion and fission factors signaling impaired muscle repair. However, chronic inflammation did not adversely affect muscles at the vaccination site or humoral immune responses; nevertheless, it partially reduced the cell-mediated immune response, particularly T-cell activation. These findings underscore the importance of addressing mRNA vaccine toxicity and immunogenicity in the context of chronic inflammation, ensuring their safe and effective utilization, particularly among vulnerable populations with immune-mediated inflammatory diseases.
ABSTRACT
DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.
Subject(s)
DNA, Mitochondrial , Transcription Activator-Like Effectors , Animals , Humans , Mice , Adenine , Cytosine , DNA, Mitochondrial/genetics , Gene Editing , RNA , Transcription Activator-Like Effectors/metabolism , Protein EngineeringABSTRACT
The E6 and E7 proteins of specific subtypes of human papillomavirus (HPV), including HPV 16 and 18, are highly associated with cervical cancer as they modulate cell cycle regulation. The aim of this study was to investigate the potential antitumor effects of a messenger RNA-HPV therapeutic vaccine (mHTV) containing nononcogenic E6 and E7 proteins. To achieve this, C57BL/6j mice were injected with the vaccine via both intramuscular and subcutaneous routes, and the resulting effects were evaluated. mHTV immunization markedly induced robust T cell-mediated immune responses and significantly suppressed tumor growth in both subcutaneous and orthotopic tumor-implanted mouse model, with a significant infiltration of immune cells into tumor tissues. Tumor retransplantation at day 62 postprimary vaccination completely halted progression in all mHTV-treated mice. Furthermore, tumor expansion was significantly reduced upon TC-1 transplantation 160 days after the last immunization. Immunization of rhesus monkeys with mHTV elicited promising immune responses. The immunogenicity of mHTV in nonhuman primates provides strong evidence for clinical application against HPV-related cancers in humans. All data suggest that mHTV can be used as both a therapeutic and prophylactic vaccine.
Subject(s)
Oncogene Proteins, Viral , Papillomavirus Infections , Papillomavirus Vaccines , Uterine Cervical Neoplasms , Humans , Female , Animals , Mice , Human Papillomavirus Viruses , Oncogene Proteins, Viral/genetics , Papillomavirus Infections/prevention & control , RNA, Messenger/genetics , Papillomavirus E7 Proteins/genetics , Mice, Inbred C57BL , Vaccination/methods , Immunization , Uterine Cervical Neoplasms/prevention & controlABSTRACT
Argyrodite-type Li6 PS5 Cl (LPSCl) has attracted much attention as a solid electrolyte for all-solid-state batteries (ASSBs) because of its high ionic conductivity and good mechanical flexibility. LPSCl, however, has challenges of translating research into practical applications, such as irreversible electrochemical degradation at the interface between LPSCl and cathode materials. Even for Li-ion batteries (LIBs), liquid electrolytes have the same issue as electrolyte decomposition due to interfacial instability. Nonetheless, current LIBs are successfully commercialized because functional electrolyte additives give rise to the formation of stable cathode-electrolyte interphase (CEI) and solid-electrolyte interphase (SEI) layers, leading to supplementing the interfacial stability between electrolyte and electrode. Herein, inspired by the role of electrolyte additives for LIBs, trimethylsilyl compounds are introduced as solid electrolyte additives for improving the interfacial stability between sulfide-based solid electrolytes and cathode materials. 2-(Trimethylsilyl)ethanethiol (TMS-SH), a solid electrolyte additive, is oxidatively decomposed during charge, forming a stable CEI layer. As a result, the CEI layer derived from TMS-SH suppresses the interfacial degradation between LPSCl and LiCoO2 , thereby leading to the excellent electrochemical performance of Li | LPSCl | LiCoO2 , such as superior cycle life over 2000 cycles (85.0% of capacity retention after 2000 cycles).
ABSTRACT
Developing new adjuvants that can effectively induce both humoral and cellular immune responses while broadening the immune response is of great value. In this study, we aimed to develop GM-CSF- or IL-18-expressing single-stranded RNA (ssRNA) adjuvants based on the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) and tested their efficacy in combination with ovalbumin (OVA) or inactivated influenza vaccines. Notably, cytokine-expressing RNA adjuvants increased the expression of antigen-presenting cell activation markers. Specifically, GM-CSF-expressing RNA adjuvants increased CD4+T cell responses, while IL-18-expressing RNA adjuvants increased CD8+T cell responses in mice when combined with OVA. In addition, cytokine-expressing RNA adjuvants increased the frequency of polyclonal T cells in combination with the influenza vaccine and reduced the clinical illness scores and weight loss of mice after viral challenge. Collectively, our results suggest that cytokine-expressing RNA adjuvants can be applied to protein-based or inactivated vaccines to increase their efficacy.
ABSTRACT
Microscopic visualization of DNA molecules is a simple, intuitive, and powerful method. Nonetheless, DNA-molecule quantification methods that employ microscopic visualization have not been reported so far. In this study, a new quantitative approach is presented that enables the counting of individual DNA molecules that have been rendered visible by fluorescence microscopy. Toward this, a microfluidic device was employed that directed DNA molecules into microchannels and deposited the molecules onto a positively charged surface. This microfluidic device had a vertically tapered channel inlet structure that prevented the accumulation of excess DNA molecules in the channel inlet while creating a tapering flow, thereby ensuring the even distribution of the DNA molecules in the microchannels. The channel heights and the density of positive charges on the surface were optimized for analysis. The linearity of this method with respect to the determination of the concentration of DNA in solutions was subsequently determined. The limit of detection was 0.48 fg/µL, which corresponds to 64 molecules of 7.25 kbp DNA in 1 µL of sample. This quantitative approach was finally used to count two types of plasmids co-transformed in an E. coli cell; a measurement that is typically considered challenging with gel electrophoresis.
Subject(s)
Microfluidic Analytical Techniques , Escherichia coli/genetics , DNA/genetics , DNA/analysis , Microscopy, Fluorescence , PlasmidsABSTRACT
Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor-enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.
Subject(s)
DNA, Mitochondrial , Gene Editing , Humans , DNA, Mitochondrial/genetics , Gene Editing/methods , Mitochondria/metabolism , Mutation , Cytosine/metabolism , CRISPR-Cas SystemsABSTRACT
We present two methods for enhancing the efficiency of mitochondrial DNA (mtDNA) editing in mice with DddA-derived cytosine base editors (DdCBEs). First, we fused DdCBEs to a nuclear export signal (DdCBE-NES) to avoid off-target C-to-T conversions in the nuclear genome and improve editing efficiency in mtDNA. Second, mtDNA-targeted TALENs (mitoTALENs) are co-injected into mouse embryos to cleave unedited mtDNA. We generated a mouse model with the m.G12918A mutation in the MT-ND5 gene, associated with mitochondrial genetic disorders in humans. The mutant mice show hunched appearances, damaged mitochondria in kidney and brown adipose tissues, and hippocampal atrophy, resulting in premature death.
Subject(s)
DNA, Mitochondrial , Mitochondrial Diseases , Animals , Cytosine , DNA, Mitochondrial/genetics , Endonucleases/metabolism , Gene Editing/methods , Humans , Mice , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Diseases/genetics , Nuclear Export Signals/genetics , Transcription Activator-Like Effector Nucleases/geneticsABSTRACT
Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.
Subject(s)
DNA, Mitochondrial , Mitochondrial Diseases , Animals , CRISPR-Cas Systems , Cytosine/metabolism , DNA, Mitochondrial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Editing , Humans , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Diseases/genetics , PurinesABSTRACT
In this paper, experimental validation of high precision web handling for a two-actuator-based roll-to-roll (R2R) system is presented. To achieve this, the tension control loop is utilized to regulate the tension in the unwinder module, and the velocity loop is utilized to regulate the web speed in the rewinder module owing to the limitation of the number of actuators. Moreover, the radius estimation algorithm is applied to achieve an accurate web speed and the control sequence of the web handling in the longitudinal axis is developed to manipulate the web handling for convenience. Having these, the tension control performances are validated within ±0.79, ±1.32 and ±1.58 percent tension tracking error and 1.6, 1.53 and 1.33 percent web speed error at the speeds of 0.1 m/s, 0.2 m/s, and 0.3 m/s, respectively. The tension control performance is verified within ±0.3 N tracking error in the changes of the reference tension profile at 0.1 m/s web speed. Lastly, the air floating roller is used to minimize the friction terms and the inertia of the idle roller in the tension zone so that tension control performance can be better achieved during web transportation.
ABSTRACT
Plant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system1, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector subarray plasmids, to assemble DddA-derived cytosine base editor (DdCBE)2 plasmids and used the resulting DdCBEs to efficiently promote point mutagenesis in mitochondria and chloroplasts. Our DdCBEs induced base editing in lettuce or rapeseed calli at frequencies of up to 25% (mitochondria) and 38% (chloroplasts). We also showed DNA-free base editing in chloroplasts by delivering DdCBE mRNA to lettuce protoplasts to avoid off-target mutations caused by DdCBE-encoding plasmids. Furthermore, we generated lettuce calli and plantlets with edit frequencies of up to 99%, which were resistant to streptomycin or spectinomycin, by introducing a point mutation in the chloroplast 16S rRNA gene.
Subject(s)
Brassica napus/genetics , Cloning, Organism/methods , DNA, Chloroplast , DNA, Mitochondrial , Gene Editing/methods , Lactuca/genetics , Plant Breeding/methods , Crops, Agricultural/geneticsABSTRACT
DddA-derived cytosine base editors (DdCBEs), composed of the split interbacterial toxin DddAtox, transcription activator-like effector (TALE), and uracil glycosylase inhibitor (UGI), enable targeted C-to-T base conversions in mitochondrial DNA (mtDNA). Here, we demonstrate highly efficient mtDNA editing in mouse embryos using custom-designed DdCBEs. We target the mitochondrial gene, MT-ND5 (ND5), which encodes a subunit of NADH dehydrogenase that catalyzes NADH dehydration and electron transfer to ubiquinone, to obtain several mtDNA mutations, including m.G12918A associated with human mitochondrial diseases and m.C12336T that incorporates a premature stop codon, creating mitochondrial disease models in mice and demonstrating a potential for the treatment of mitochondrial disorders.
Subject(s)
DNA, Mitochondrial/genetics , Gene Editing/methods , Genes, Mitochondrial/genetics , Animals , Electron Transport , Female , Male , Mice , Mice, Inbred C57BL , Mitochondria , Mitochondrial Diseases/genetics , Mitochondrial Proteins/genetics , Mutation , NADH Dehydrogenase/genetics , NIH 3T3 Cells , Transcription Activator-Like Effectors/geneticsABSTRACT
DNA binding fluorescent proteins are useful probes for a broad range of biological applications. Fluorescent protein (FP)-tagging allows DNA binding proteins expressed within a living cell to be directly visualised, in real-time, to study DNA binding patterns and dynamics. Moreover, FP-tagged DNA binding proteins (FP-DBP) have allowed the imaging of single proteins bound to large elongated DNA molecules with a fluorescence microscope. Although there are numerous DNA binding proteins, only a small portion of them have been exploited to construct FP-DBPs to study molecular motion in a cell or in vitro single-molecule visualisation. Therefore, it would be informative to review FP-DBP for further development. Here, we summarise the design of FP-DBPs and their brightness, photostability, pKa, maturation rate, and binding affinity (Kd) characteristics. Then, we review the applications of FP-DBP in cells to study chromosome dynamics, DNA replication, transcription factors, DNA damage, and repair. Finally, we focus on single DNA molecule visualisation using FP-DBP.
Subject(s)
DNA-Binding Proteins/chemistry , DNA/metabolism , Fluorescent Dyes/chemistry , Luminescent Proteins/chemistry , Animals , Cell Line , Chromosomes/metabolism , DNA/analysis , DNA Damage/physiology , DNA Repair/physiology , DNA Replication/physiology , DNA-Binding Proteins/metabolism , Fluorescent Dyes/metabolism , Humans , Luminescent Proteins/metabolism , Microscopy/methods , Mitosis/physiology , Plants , Protein Binding , Single-Cell Analysis/methodsABSTRACT
Although carbon nanotubes (CNTs) are remarkable materials with many exceptional characteristics, their poor chemical functionality limits their potential applications. Herein, a strategy is proposed for functionalizing CNTs, which can be achieved with any functional group (FG) without degrading their intrinsic structure by using a deoxyribonucleic acid (DNA)-binding peptide (DBP) anchor. By employing a DBP tagged with a certain FG, such as thiol, biotin, and carboxyl acid, it is possible to introduce any FG with a controlled density on DNA-wrapped CNTs. Additionally, different types of FGs can be introduced on CNTs simultaneously, using DBPs tagged with different FGs. This method can be used to prepare CNT nanocomposites containing different types of nanoparticles (NPs), such as Au NPs, magnetic NPs, and quantum dots. The CNT nanocomposites decorated with these NPs can be used as reusable catalase-like nanocomposites with exceptional catalytic activities, owing to the synergistic effects of all the components. Additionally, the unique DBP-DNA interaction allows the on-demand detachment of the NPs attached to the CNT surface; further, it facilitates a CNT chirality-specific NP attachment and separation using the sequence-specific programmable characteristics of oligonucleotides. The proposed method provides a novel chemistry platform for constructing new functional CNTs suitable for diverse applications.
Subject(s)
Nanocomposites , Nanotubes, Carbon , Peptides , DNA/metabolism , Nanocomposites/chemistry , Nanotubes, Carbon/chemistry , Peptides/chemistry , Peptides/metabolism , Quantum DotsABSTRACT
The tunnel field-effect transistor (TFET) with surrounding channel nanowire (SCNW) structure promises better performance than the conventional planar TFET in terms of subthreshold swing (SS) and on-current (ION). In spite of the advantages of SCNW TFET, there are some technical issues in the aspects of a hump phenomenon in subthreshold region and a high ambipolar current (IAMB) in off-state. In order to overcome these issues, a novel dual-gate SCNW TFET (DG-SCNW TFET) with differential gate work functions (WFs) and a gate-drain underlap is proposed and studied by using technology computer-aided design (TCAD) simulation. In addition, a hetero-junction with SiGe source is applied to improve the device performance. Finally, it is confirmed that the optimized DG-SCNW TFET shows the remarkable performance comparing with the control device.
ABSTRACT
Large DNA molecules are a promising platform for in vitro single-molecule biochemical analysis to investigate DNA-protein interactions by fluorescence microscopy. For many studies, intercalating fluorescent dyes have been primary DNA staining reagents, but they often cause photo-induced DNA breakage as well as structural deformation. As a solution, we previously developed several fluorescent-protein DNA-binding peptides or proteins (FP-DBP) for reversibly staining DNA molecules without structural deformation or photo-induced damage. However, they cannot stain DNA in a condition similar to a physiological salt concentration that most biochemical reactions require. Given these concerns, here we developed a salt-tolerant FP-DBP: truncated transcription activator-like effector (tTALE-FP), which can stain DNA up to 100 mM NaCl. Moreover, we found an interesting phenomenon that the tTALE-FP stained DNA evenly in 1 × TE buffer but showed AT-rich specific patterns from 40 mM to 100 mM NaCl. Using an assay based on fluorescence resonance energy transfer, we demonstrated that this binding pattern is caused by a higher DNA binding affinity of tTALE-FP for AT-rich compared to GC-rich regions. Finally, we used tTALE-FP in a single molecule fluorescence assay to monitor real-time restriction enzyme digestion of single DNA molecules. Altogether, our results demonstrate that this protein can provide a useful alternative as a DNA stain over intercalators.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/chemistry , DNA/metabolism , Fluorescent Dyes/chemistry , Intercalating Agents/metabolism , Staining and Labeling/methods , Transcription Activator-Like Effectors/metabolism , DNA-Binding Proteins/chemistry , Fluorescence , Fluorescence Resonance Energy Transfer , Humans , Intercalating Agents/chemistry , Microscopy, Fluorescence , Single Molecule Imaging/methods , Transcription Activator-Like Effectors/chemistryABSTRACT
BACKGROUND AND OBJECTIVES: Noise levels and room acoustic parameters at a tertiary referral hospital, Seoul National University Hospital (SNUH) in Korea, are investigated. MATERIALS AND METHODS: Through a questionnaire, acoustically problematic rooms are identified. Noise levels in emergency rooms (ERs) and intensive care units (ICUs) are measured over about three days. Acoustically critical and problematic rooms in the otolaryngology department are measured including examination rooms, operating rooms, nurse stations, receptions, and patient rooms. RESULTS: The A-weighted equivalent noise level, LAeq, ranges from 54 to 56 dBA, which is at least 10 dB lower than the noise levels of 65 to 73 dBA measured in American ERs. In an ICU, the noise level for the first night was 66 dBA, which came down to 56 dBA for the next day. The noise levels during three different ear surgeries vary from 57 to 62 dBA, depending on the use of surgical drills and suctions. The noise levels in a patient room is found to be 47 dBA, while the nurse stations and the receptions have high noise levels up to 64 dBA. The reverberation times in an operation room, examination room, and single patient room are found to be below 0.6 s. CONCLUSIONS: At SNUH, the nurse stations and receptions were found to be quite noisy. The ERs were quieter than in the previous studies. The measured reverberation times seemed low enough but some other nurse stations and examination rooms were not satisfactory according to the questionnaire.
ABSTRACT
The recent advances in the single cell genome analysis are generating a considerable amount of novel insights into complex biological systems. However, there are still technical challenges because each cell has a single copy of DNA to be amplified in most single cell genome analytical methods. In this paper, we present a novel approach to directly visualize a genomic map on a large DNA molecule instantly stained with red and green DNA-binding fluorescent proteins without DNA amplification. For this visualization, we constructed a few types of fluorescent protein-fused DNA-binding proteins: H-NS (histone-like nucleoid-structuring protein), DNA-binding domain of BRCA1 (breast cancer 1), high mobility group-1 (HMG), and lysine tryptophan (KW) repeat motif. Because H-NS and HMG preferentially bind A/T-rich regions, we combined A/T specific binder (H-NS-mCherry and HMG-mCherry as red color) and a non-specific complementary DNA binder (BRCA1-eGFP and 2(KW)2-eGFP repeat as green color) to produce a sequence-specific two-color DNA physical map for efficient optical identification of single DNA molecules.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/analysis , Green Fluorescent Proteins/metabolism , Image Processing, Computer-Assisted/methods , Microscopy, Fluorescence/methods , Single-Cell Analysis/methods , DNA/chemistry , DNA/metabolism , HumansABSTRACT
Fluorophore-linked, sequence-specific DNA binding reagents can visualize sequence information on a large DNA molecule. In this paper, we synthesized newly designed TAMRA-linked polypyrrole to visualize adenine and thymine base pairs. A fluorescent image of the stained DNA molecule generates an intensity profile based on A/T frequency, revealing a characteristic sequence composition pattern. Computer-aided comparison of this intensity pattern with the genome sequence allowed us to determine the DNA sequence on a visualized DNA molecule from possible intensity profile pattern candidates for a given genome. Moreover, TAMRA-polypyrrole offers robust advantages for single DNA molecule detection: no fluorophore-mediated photocleavage and no structural deformation, since it exhibits a sequence-specific pattern alone without the use of intercalating dyes such as YOYO-1. Accordingly, we were able to identify genomic DNA fragments from Escherichia coli cells by aligning them to the genomic A/T frequency map based on TAMRA-polypyrrole-generated intensity profiles. Furthermore, we showed band and interband patterns of polytene chromosomal DNA stained with TAMRA-polypyrrole because it prefers to bind AT base pairs.