Standards for Quantitative Measurement of DNA Damage in Mammalian Cells.

As the potential applications of DNA diagnostics continue to expand, there is a need for improved methods and standards for DNA analysis. This report describes several methods that could be considered for the production of reference materials for the quantitative measurement of DNA damage in mammalian cells. With the focus on DNA strand breaks, potentially useful methods for assessing DNA damage in mammalian cells are reviewed. The advantages and limitations of each method, as well as additional concerns with respect to reference material development, are also discussed. In conclusion, we outline strategies for developing candidate DNA damage reference materials that could be adopted by research laboratories in a wide variety of applications.

Quantifying engineered nanomaterial toxicity: comparison of common cytotoxicity and gene expression measurements.

BACKGROUND: When evaluating the toxicity of engineered nanomaterials (ENMS) it is important to use multiple bioassays based on different mechanisms of action. In this regard we evaluated the use of gene expression and common cytotoxicity measurements using as test materials, two selected nanoparticles with known differences in toxicity, 5 nm mercaptoundecanoic acid (MUA)-capped InP and CdSe quantum dots (QDs). We tested the effects of these QDs at concentrations ranging from 0.5 to 160 µg/mL on cultured normal human bronchial epithelial (NHBE) cells using four common cytotoxicity assays: the dichlorofluorescein assay for reactive oxygen species (ROS), the lactate dehydrogenase assay for membrane viability (LDH), the mitochondrial dehydrogenase assay for mitochondrial function, and the Comet assay for DNA strand breaks. RESULTS: The cytotoxicity assays showed similar trends when exposed to nanoparticles for 24 h at 80 µg/mL with a threefold increase in ROS with exposure to CdSe QDs compared to an insignificant change in ROS levels after exposure to InP QDs, a twofold increase in the LDH necrosis assay in NHBE cells with exposure to CdSe QDs compared to a 50% decrease for InP QDs, a 60% decrease in the mitochondrial function assay upon exposure to CdSe QDs compared to a minimal increase in the case of InP and significant DNA strand breaks after exposure to CdSe QDs compared to no significant DNA strand breaks with InP. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) data for cells exposed for 6 h at a concentration of 80 µg/mL were consistent with the cytotoxicity assays showing major differences in DNA damage, DNA repair and mitochondrial function gene regulatory responses to the CdSe and InP QDs. The BRCA2, CYP1A1, CYP1B1, CDK1, SFN and VEGFA genes were observed to be upregulated specifically from increased CdSe exposure and suggests their possible utility as biomarkers for toxicity. CONCLUSIONS: This study can serve as a model for comparing traditional cytotoxicity assays and gene expression measurements and to determine candidate biomarkers for assessing the biocompatibility of ENMs.

Copper oxide nanoparticle mediated DNA damage in terrestrial plant models.

Engineered nanoparticles, due to their unique electrical, mechanical, and catalytic properties, are presently found in many commercial products and will be intentionally or inadvertently released at increasing concentrations into the natural environment. Metal- and metal oxide-based nanomaterials have been shown to act as mediators of DNA damage in mammalian cells, organisms, and even in bacteria, but the molecular mechanisms through which this occurs are poorly understood. For the first time, we report that copper oxide nanoparticles induce DNA damage in agricultural and grassland plants. Significant accumulation of oxidatively modified, mutagenic DNA lesions (7,8-dihydro-8-oxoguanine; 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 4,6-diamino-5-formamidopyrimidine) and strong plant growth inhibition were observed for radish (Raphanus sativus), perennial ryegrass (Lolium perenne), and annual ryegrass (Lolium rigidum) under controlled laboratory conditions. Lesion accumulation levels mediated by copper ions and macroscale copper particles were measured in tandem to clarify the mechanisms of DNA damage. To our knowledge, this is the first evidence of multiple DNA lesion formation and accumulation in plants. These findings provide impetus for future investigations on nanoparticle-mediated DNA damage and repair mechanisms in plants.

Development of a Reference Method and Materials for Quantitative Measurement of UV-Induced DNA Damage in Mammalian Cells: Comparison of Comet Assay and Cell Viability.

Application of DNA damage diagnostic tests is rapidly growing, in particular for ovarian, prostate, and skin cancers; environmental monitoring; chronic and degenerative diseases; and male infertility. Such tests suffer from significant variability among different laboratories due the lack of standardization, experimental validation, and differences in data interpretation. Reference methods and materials for quantitative measurement of UVA-induced DNA damage in mammalian cells are frequently needed. In this study, we examined the use of the single-cell gel electrophoresis (comet) assay to assess the UVA-induced DNA damage in surface-attached Chinese hamster ovary (CHO) cells treated with a photosensitizer as a candidate cellular oxidative damage reference material. We found that the comet images became diffused and the viability of the cells decreased substantially (>20%) as the UVA dose and benzo [a] pyrene (BaP) concentration exceeded 6.3 J/cm2 and 10-6 mol/L BaP. Maintaining the conditions of exposure within this range can improve DNA damage measurement fidelity, particularly if used as a quantitative reference method and to produce materials considered as an in vitro standard for the comet assay.

Genotoxic Effects of Etoposide, Bleomycin, and Ethyl Methanesulfonate on Cultured CHO Cells: Analysis by GC-MS/MS and Comet Assay.

To evaluate methods for analysis of genotoxic effects on mammalian cell lines, we tested the effect of three common genotoxic agents on Chinese hamster ovary (CHO) cells by single-cell gel electrophoresis (comet assay) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Suspension-grown CHO cells were separately incubated with etoposide, bleomycin, and ethyl methanesulfonate and analyzed by an alkaline comet assay and GC-MS/MS. Although DNA strand breaks were detected by the comet assay after treatment with all three agents, GC-MS/MS could only detect DNA nucleobase lesions oxidatively induced by bleomycin. This demonstrates that although GC-MS/MS has limitations in detection of genotoxic effects, it can be used for selected chemical genotoxins that contribute to oxidizing processes. The comet assay, used in combination with GC-MS/MS, can be a more useful approach to screen a wide range of chemical genotoxins as well as to monitor other DNA-damaging factors.

Cellular Reference Materials for DNA Damage Using Electrochemical Oxidation.

Reference materials are needed to quantify the level of DNA damage in cells, to assess sources of measurement variability and to compare results from different laboratories. The comet assay (single cell gel electrophoresis) is a widely used method to determine DNA damage in the form of strand breaks. Here we examine the use of electrochemical oxidation to produce DNA damage in cultured mammalian cells and quantify its percentage using the comet assay. Chinese hamster ovary (CHO) cells were grown on an indium tin oxide electrode surface and exposed 12 h to electrochemical potentials ranging from 0.5 V to 1.5 V (vs Ag/AgCl). The resulting cells were harvested and analyzed by comet and a cell viability assay. We observed a linear increase in the percentage (DNA in tail) of strand breaks along with a loss of cell viability with increasing oxidation potential value. The results indicate that electrochemically induced DNA damage can be produced in mammalian cells under well-controlled conditions and could be considered in making a cellular reference material for the comet assay.

Controlled potential electro-oxidation of genomic DNA.

Exposure of mammalian cells to oxidative stress can result in DNA damage that adversely affects many cell processes. Lack of dependable DNA damage reference materials and standardized measurement methods, despite many case-control studies hampers the wider recognition of the link between oxidatively degraded DNA and disease risk. We used bulk electrolysis in an electrochemical system and gas chromatographic mass spectrometric analysis (GC/MS/MS) to control and measure, respectively, the effect of electrochemically produced reactive oxygen species on calf thymus DNA (ct-DNA). DNA was electro-oxidized for 1 h at four fixed oxidizing potentials (E = 0.5 V, 1.0 V, 1.5 V and 2 V (vs Ag/AgCl)) using a high surface area boron-doped diamond (BDD) working electrode (WE) and the resulting DNA damage in the form of oxidatively-modified DNA lesions was measured using GC/MS/MS. We have shown that there are two distinct base lesion formation modes in the explored electrode potential range, corresponding to 0.5 V < E < 1.5 V and E > 1.5 V. Amounts of all four purine lesions were close to a negative control levels up to E = 1.5 V with evidence suggesting higher levels at the lowest potential of this range (E = 0.5 V). A rapid increase in all base lesion yields was measured when ct-DNA was exposed at E = 2 V, the potential at which hydroxyl radicals were efficiently produced by the BDD electrode. The present results demonstrate that controlled potential preparative electrooxidation of double-stranded DNA can be used to purposely increase the levels of oxidatively modified DNA lesions in discrete samples. It is envisioned that these DNA samples may potentially serve as analytical control or quality assurance reference materials for the determination of oxidatively induced DNA damage.

Real-time telomerase assay of less-invasively collected esophageal cell samples.

Genomic and proteomic efforts have discovered a complex list of biomarkers that identify human disease, stratify risk of disease within populations, and monitor drug or therapy responses for treatment. Attention is needed to characterize these biomarkers and to develop high-throughput technologies to evaluate their accuracy and precision. Telomerase activity is correlated with tumor progression, indicating cells that express telomerase possess aggressive clinical behavior and that telomerase activity could be a clinically important cancer biomarker. Traditionally, the detection of cancer has involved invasive procedures to procure samples. There is a need for less invasive approaches suitable for population- and clinic-based assays for cancer early detection. Esophageal balloon cytology (EBC) is a low-invasive screening technique, which samples superficial epithelial cells from the esophagus. Since telomerase activity is absent in superficial cells of normal esophageal squamous epithelium but is often present in superficial cells from dysplastic lesions and ESCCs, measuring telomerase activity in EBC samples may be a good way to screen for these lesions. The development of rapid real-time telomerase activity assays raises the possibility of extending such screening to high-risk populations. In this study, we evaluate the feasibility of using rapid Real-Time Telomerase Repeat Amplification Protocol (RTTRAP) for the analysis of NIST telomerase candidate reference material and esophageal clinical samples. The telomerase activity of eight EBC samples was also measured by capillary electrophoresis of RTTRAP products, RApidTRAP, and hTERT mRNA RT-PCR assays. These findings demonstrate the feasibility of using the RTTRAP assay in EBC samples and suggest that individuals from high-risk populations can be screened for telomerase activity.

The Comet Assay: Automated Imaging Methods for Improved Analysis and Reproducibility.

Sources of variability in the comet assay include variations in the protocol used to process the cells, the microscope imaging system and the software used in the computerized analysis of the images. Here we focus on the effect of variations in the microscope imaging system and software analysis using fixed preparations of cells and a single cell processing protocol. To determine the effect of the microscope imaging and analysis on the measured percentage of damaged DNA (% DNA in tail), we used preparations of mammalian cells treated with etoposide or electrochemically induced DNA damage conditions and varied the settings of the automated microscope, camera, and commercial image analysis software. Manual image analysis revealed measurement variations in percent DNA in tail as high as 40% due to microscope focus, camera exposure time and the software image intensity threshold level. Automated image analysis reduced these variations as much as three-fold, but only within a narrow range of focus and exposure settings. The magnitude of variation, observed using both analysis methods, was highly dependent on the overall extent of DNA damage in the particular sample. Mitigating these sources of variability with optimal instrument settings facilitates an accurate evaluation of cell biological variability.

Analytical validation of telomerase activity for cancer early detection: TRAP/PCR-CE and hTERT mRNA quantification assay for high-throughput screening of tumor cells.

Activation of telomerase plays a critical role in unlimited proliferation and immortalization of cells. Telomerase activity has been shown to correlate with tumor progression, indicating that tumors expressing this enzyme possess aggressive clinical behavior and that telomerase activity may be a useful biomarker for early detection of cancer. However, measurements of telomerase activity by current methods such as telomeric repeat amplification protocol (TRAP)/polymerase chain reaction (PCR) or antibody-based radioimmunoassay (RIA) are low-throughput and not robust enough to easily accommodate the required statistical analysis to determine whether telomerase activity is a practical biomarker. As part of the National Cancer Institute Early Detection Research Network of analytical validation, we have developed a robot assisted TRAP assay (RApidTRAP) of telomerase, a potential biomarker for cancer early detection. Measurements of human telomerase reverse transcriptase catalytic subunit (hTERT) mRNA were performed in concert with measurement of telomerase activity. For this purpose we determined hTERT mRNA concentration and telomerase activity in human normal (RPE-28) and cancer (A549) cell lines as well as in human serum (SRM 1951A). Telomerase activity measurements were made using the TRAP/PCR capillary electrophoresis (CE) method on (50 to 1000) cells/reaction isolated from cell extracts. Measurement of hTERT mRNA was made using specific primers and probes on a LightCycler in the range of (10 to 7000) cells/reaction. Comparison of high-throughput telomerase activity measurements using the robot and those performed manually were consistent in sensitivity and reproducibility. Using this combination of telomerase activity and hTERT mRNA measurements, the automated system improved efficiency over traditional TRAP/PCR methods.

Measurement of DNA biomarkers for the safety of tissue-engineered medical products, using artificial skin as a model.

To test the hypothesis that the process of tissue engineering introduces genetic damage to tissue-engineered medical products, we employed the use of five state-of-the-art measurement technologies to measure a series of DNA biomarkers in commercially available tissue-engineered skin as a model. DNA was extracted from the skin and compared with DNA from cultured human neonatal control cells (dermal fibroblasts and epidermal keratinocytes) and adult human fibroblasts from a 55-year-old donor and a 96-year-old donor. To determine whether tissue engineering caused oxidative DNA damage, gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution mass spectrometry were used to measure six oxidatively modified DNA bases as biomarkers. Normal endogenous levels of the modified DNA biomarkers were not elevated in tissue-engineered skin when compared with control cells. Next, denaturing high-performance liquid chromatography and capillary electrophoresis-single strand conformation polymorphism were used to measure genetic mutations. Specifically, the TP53 tumor suppressor gene was screened for mutations, because it is the most commonly mutated gene in skin cancer. The tissue-engineered skin was found to be free of TP53 mutations at the level of sensitivity of these measurement technologies. Lastly, fluorescence in situ hybridization was employed to measure the loss of Y chromosome, which is associated with excessive cell passage and aging. Loss of Y chromosome was not detected in the tissue-engineered skin and cultured neonatal cells used as controls. In this study, we have demonstrated that tissue engineering (for TestSkin II) does not introduce genetic damage above the limits of detection of the state-of-the-art technologies used. This work explores the standard for measuring genetic damage that could be introduced during production of novel tissue-engineered products. More importantly, this exploratory work addresses technological considerations that need to be addressed in order to expedite accurate and useful international reference standards for the emerging tissue-engineering industry.

Use of TP53 reference materials to validate mutations in clinical tissue specimens by single-strand conformational polymorphism analysis.

BACKGROUND: As genetic information moves from basic research laboratories in to the clinical testing environment, there is a critical need for reliable reference materials for the quality assurance of genetic tests. A panel of 12 plasmid clones containing wild-type or point mutations within exons 5-9 have been developed as reference materials for the detection of TP53 mutations. AIM: The goal of this study was to validate the reference materials in providing quality assurance for the detection of TP53 mutations in clinical specimens. METHODS: We studied 33 gynecological samples, 11 apparently normal samples and 22 malignant tumors of various origins. Mutations were identified using single-strand conformational polymorphism analysis with both slab gel and capillary electrophoresis. All DNA samples were amplified with fluorescently labeled PCR primers specific for exons 5-9 for mutation detection. RESULTS: Of the 33 patient samples tested, mutations and polymorphisms were found in six specimens in three of the five exons scanned; no mutations were found in exons 7 or 9. Both a mutation and polymorphism were found in non-malignant specimens from the control group. The mutations were confirmed by DNA sequence analysis of the regions scanned. CONCLUSIONS: Mutations and polymorphisms were detected in the clinical samples. All of the mutations were silent except for one non-conservative mutation in exon 5, codon 181. This study demonstrates the usefulness of the National Institute of Standards and Technology (NIST) TP53 reference panel in TP53 mutation detection in clinical tissue specimens.

Renewable standard reference material for the detection of TP53 mutations.

BACKGROUND: Numerous DNA-based tests are currently in use or under development for the detection of mutations associated with disease. Most of the current methods use PCR amplification technologies and detection after separation or chromatography of the products. We have developed a panel of standard reference materials consisting of 12 plasmid clones containing a 2.0 kb region of the TP53 gene, including exons 5-9. Eleven of these clones contain a single mutation within the mutational hot spots of the TP53 gene, the twelfth is wild-type in this region of the gene. The mutations are amino acid (aa) 128: C to T; aa 175: G to A; aa 237: T to C; aa 245: G to A; aa 248: C to T; aa 248: G to A; aa 249: G to T; aa 273: C to T; aa 273: G to A; aa 282: C to T; and aa 328: T to C. These standard reference materials (SRMs), created by site-directed mutagenesis of wild-type TP53 from a human cell line, include the specific mutations most commonly found to be associated with cancer. Their use will improve disease detection by serving as validation materials to monitor errors in measurement methods, including PCR amplification, amplicon separation, and data analysis from different technology platforms. METHODS AND RESULTS: The single point mutations of the panel were validated by capillary electrophoresis single-strand conformational polymorphism analysis, denaturing gradient gel electrophoresis, and denaturing high-performance liquid chromatography, as well as full sequence analysis of both DNA strands of the cloned material. For both heteroduplex analysis methods, the presence of the mutations was resolved for each SRM. CONCLUSION: The generation of a standard TP53 reference panel and demonstration that the panel can successfully validate mutation detection across different mutation scanning technology platforms. Hence, this panel functions as an SRM to normalize results obtained from different laboratories using different techniques.

NIST gold nanoparticle reference materials do not induce oxidative DNA damage.

One primary challenge in nanotoxicology studies is the lack of well-characterised nanoparticle reference materials which could be used as positive or negative nanoparticle controls. The National Institute of Standards and Technology (NIST) has developed three gold nanoparticle (AuNP) reference materials (10, 30 and 60 nm). The genotoxicity of these nanoparticles was tested using HepG2 cells and calf-thymus DNA. DNA damage was assessed based on the specific and sensitive measurement of four oxidatively-modified DNA lesions (8-hydroxy-2´-deoxyguanosine, 8-hydroxy-2´-deoxyadenosine, (5´S)-8,5´-cyclo-2´-deoxyadenosine and (5´R)-8,5´-cyclo-2´-deoxyadenosine) using liquid chromatography/tandem mass spectrometry. Significantly elevated, dose-dependent DNA damage was not detected at concentrations up to 0.2 µg/ml, and free radicals were not detected using electron paramagnetic resonance spectroscopy. These data suggest that the NIST AuNPs could potentially serve as suitable negative-control nanoparticle reference materials for in vitro and in vivo genotoxicity studies. NIST AuNPs thus hold substantial promise for improving the reproducibility and reliability of nanoparticle genotoxicity studies.

Manganese-induced oxidative DNA damage in neuronal SH-SY5Y cells: attenuation of thymine base lesions by glutathione and N-acetylcysteine.

Manganese (Mn) is an essential trace element required for normal function and development. However, exposure to this metal at elevated levels may cause manganism, a progressive neurodegenerative disorder with neurological symptoms similar to idiopathic Parkinson's disease (IPD). Elevated body burdens of Mn from exposure to parental nutrition, vapors in mines and smelters and welding fumes have been associated with neurological health concerns. The underlying mechanism of Mn neurotoxicity remains unclear. Accordingly, the present study was designed to investigate the toxic effects of Mn(2+) in human neuroblastoma SH-SY5Y cells. Mn(2+) caused a concentration dependent decrease in SH-SY5Y cellular viability compared to controls. The LD50 value was 12.98 µM Mn(2+) (p<0.001 for control vs. 24h Mn treatment). Both TUNEL and annexin V/propidium iodide (PI) apoptosis assays confirmed the induction of apoptosis in the cells following exposure to Mn(2+) (2 µM, 62 µM or 125 µM). In addition, Mn(2+) induced both the formation and accumulation of DNA single strand breaks (via alkaline comet assay analysis) and oxidatively modified thymine bases (via gas chromatography/mass spectrometry analysis). Pre-incubation of the cells with characteristic antioxidants, either 1mM N-acetylcysteine (NAC) or 1mM glutathione (GSH) reduced the level of DNA strand breaks and the formation of thymine base lesions, suggesting protection against oxidative cellular damage. Our findings indicate that (1) exposure of SH-SY5Y cells to Mn promotes both the formation and accumulation of oxidative DNA damage, (2) SH-SY5Y cells with accumulated DNA damage are more likely to die via an apoptotic pathway and (3) the accumulated levels of DNA damage can be abrogated by the addition of exogenous chemical antioxidants. This is the first known report of Mn(2+)-induction and antioxidant protection of thymine lesions in this SH-SY5Y cell line and contributes new information to the potential use of antioxidants as a therapeutic strategy for protection against Mn(2+)-induced oxidative DNA damage.

Functionalization-dependent induction of cellular survival pathways by CdSe quantum dots in primary normal human bronchial epithelial cells.

Quantum dots (QDs) are semiconductor nanocrystals exhibiting unique optical properties that can be exploited for many practical applications ranging from photovoltaics to biomedical imaging and drug delivery. A significant number of studies have alluded to the cytotoxic potential of these materials, implicating Cd-leaching as the causal factor. Here, we investigated the role of heavy metals in biological responses and the potential of CdSe-induced genotoxicity. Our results indicate that, while negatively charged QDs are relatively noncytotoxic compared to positively charged QDs, the same does not hold true for their genotoxic potential. Keeping QD core composition and size constant, 3 nm CdSe QD cores were functionalized with mercaptopropionic acid (MPA) or cysteamine (CYST), resulting in negatively or positively charged surfaces, respectively. CYST-QDs were found to induce significant cytotoxicity accompanied by DNA strand breakage. However, MPA-QDs, even in the absence of cytotoxicity and reactive oxygen species formation, also induced a high number of DNA strand breaks. QD-induced DNA damage was confirmed by identifying the presence of p53 binding protein 1 (53BP1) in the nuclei of exposed cells and subsequent diminishment of p53 from cytoplasmic cellular extracts. Further, high-throughput real-time PCR analyses revealed upregulation of DNA damage and response genes and several proinflammatory cytokine genes. Most importantly, transcriptome sequencing revealed upregulation of the metallothionein family of genes in cells exposed to MPA-QDs but not CYST-QDs. These data indicate that cytotoxic assays must be supplemented with genotoxic analyses to better understand cellular responses and the full impact of nanoparticle exposure when making recommendations with regard to risk assessment.

Standards for immunohistochemical imaging: a protein reference device for biomarker quantitation.

We are developing a reference device to be used in the validation of immunohistochemical imaging of biomarkers by microscopy. The prototype device consists of p53 protein immobilized at various concentrations on a glass slide. The device is designed as a reference control to be used with assays that incorporate commercially available anti-p53 antibodies. p53 protein was characterized by mass spectrometry and covalently immobilized through amide linkage to the (3-aminopropyl)trietoxysilane-modified glass surface. This procedure is reproducible and provides a chemically stable product in high yield. The surface-bound protein was shown to be immunoreactive by its specific interaction with anti-p53 antibody (Ab) and detection by absorbance and fluorescence spectroscopy. Also, comparison was made with microscopic images of Ab-stained tissue samples, known to stain positive for p53. Further development will be required to establish accurate surface protein concentrations in the range required for specific clinical applications.

High-throughput DNA diagnostic measurements using capillary electrophoresis: p53, fragile X and telomerase.

Capillary electrophoresis (CE) has become recognized as a powerful tool for the characterization of DNA. It has numerous advantages over slab-gel electrophoresis in that it is fast, highly reproducible and easy to automate. It is well known for its contribution to success in sequencing the human genome, but it is equally important in a wide range of forensic and pharmaceutical applications. Of these applications, CE plays a large and important role in mutation scanning and DNA sizing. From the author's laboratory, three previously published examples are given of clinical applications in this area that have benefited from the use of capillary electrophoresis: the detection of p53 mutations by single strand conformational polymorphism, the analysis of fragile X syndrome and the measurement of telomerase activity. There are many examples from other laboratories where CE has played an important role in this field. For acceptance by the medical community, there must be a clear demonstration that capillary electrophoresis can replace and improve previous slab-gel methods. In this regard, the examples given in this review help to demonstrate that CE can replace previous slab-gel methods and show that CE can improve a wide range of applications in the medical field.

Conformational analysis of the telomerase RNA pseudoknot hairpin by Raman spectroscopy.

We have measured the temperature-dependent Raman spectra of two 30-mer ribonucleotides that represent the wild-type (WT) and dyskeratosis congenita (DKC) mutant (MT) GC (107-108) --> AG structures of the pseudoknot hairpin region of human telomerase RNA. We have used these structures, previously characterized by UV-melting and NMR, as a model system for our Raman investigation. We observe that Raman hypochromism of vibrational bands, previously assigned to specific bases or conformational RNA markers, reflect temperature-dependent alterations in the pentaloop and stem structures of these two oligonucleotides. We also observe that the intense nu(s)(O-P-O) band at 812 cm(-1) indicates the presence of A-form backbone structure at relatively low temperatures in both the WT and MT RNA sequences. The mutation induces a decrease in the intensity of the uridine (rU) band at 1244 cm(-1) associated with C2'-endo/anti ribose conformation in the pentaloop. Two transition temperatures (T(m) ) were determined from the analysis of Raman difference intensity-temperature profiles of the 1256 cm(-1) band, which is associated with vibrations of cytidine (rC) residues, in particular, the C2'-endo/anti ribose conformation (T(m) 1 = 23.6 +/- 1.6 degrees C for WT and 19.7 +/- 2.8 degrees C for MT; T(m) 2 = 68.9 +/- 1.8 degrees C for WT and 70.9 +/- 1.1 degrees C for MT). From these results we can conclude that the DKC mutant 30-mer exhibits a lower stability in the pentaloop region and a slightly higher stability in the stem region than the WT 30-mer. This demonstrates that Raman bands, previously assigned to specific bases or conformational RNA markers, can be used to probe local structural features of the telomerase pseudoknot hairpin sequence.