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PubMed2026

Pan-cancer analysis identifies GPRIN1 as a prognostic biomarker and promoter of cell proliferation in pancreatic cancer.

BACKGROUND: G protein-regulated inducer of neurite outgrowth 1 (GPRIN1), an emerging modulator of GPCR signaling, has been implicated in oncogenesis. However, its comprehensive role across human cancers, particularly in reshaping the tumor microenvironment (TME), remains poorly characterized. We aimed to elucidate the pan-cancer significance of GPRIN1 in TME modulation and its therapeutic implications. METHODS: We analyzed multi-omics data from TCGA and other public databases, performing a systematic analysis of GPRIN1 regarding expression, prognosis, immune infiltration, and genomic instability across 33 cancer types. To validate these bioinformatic findings, we performed lentiviral shRNA-mediated knockdown in pancreatic (PANC-1) and hepatic (HepG2) cancer cells to assess proliferation and migration. Crucially, the clinical relevance of GPRIN1 was further validated in an independent cohort of pancreatic cancer patients (N = 17) using immunohistochemistry (IHC). RESULTS: The analysis identified a lineage-dependent expression pattern. Epithelial tumors exhibited upregulation, whereas glioblastoma samples displayed downregulation. GPRIN1 expression consistently correlated with immune subtypes and CD8+ T cell abundance. In vitro assays demonstrated that GPRIN1 depletion significantly inhibited cell proliferation and migration (P < 0.0001). In the clinical validation cohort, multivariate Cox regression analysis identified high GPRIN1 protein levels as an independent predictor of post-operative recurrence. These patients also showed a trend toward extended overall survival. CONCLUSIONS: These findings define GPRIN1 as a context-dependent regulator of the TME. By integrating computational and experimental data, this study supports GPRIN1 as a potential biomarker for risk assessment in pancreatic cancer.

Humans↗

PubMed2004

Mechanisms of failure of total hip replacements: lessons learned from retrieval studies.

The value of implant retrieval analysis in orthopaedic surgery has been well recognized. Prosthetic devices retrieved for cause at revision surgery (for implant failure) or devices retrieved postmortem from patients with clinically successful reconstructions provide a unique set of specimens that can be studied to evaluate the effect of the implant on the host environment and the effect of the host environment on the implant. A systematic analysis of retrieved components, in combination with histologic, radiographic, and clinical data can provide valuable insights into the mechanisms of failure of the biomaterials used in joint replacement applications. From the hip implant retrieval studies reported to date, it has been established that the local reaction to particulate wear debris initiates the formation of a granulomatous tissue that ultimately invades the bone-implant interface and results in aseptic loosening. Cement mantle defects, noncircumferential porous coatings, and screw holes can serve as preferential access pathways for the progression of this granulomatous process yielding distinctive patterns of implant loosening and osteolysis. Continued surveillance of retrieved devices is strongly recommended to deepen our understanding of implant failure mechanisms and to evaluate the impact of newer designs and materials on the performance of joint replacement devices.

Animals↗

PubMed2006

Thyroid surgery: changing patterns of practice.

OBJECTIVE: The practice of thyroidectomy has evolved over the past 10 years with the introduction of minimally invasive surgery, laryngeal nerve monitoring, and outpatient surgery. We sought to investigate corresponding trends in the disciplines performing thyroid surgery. METHODS AND MATERIALS: The authors conducted a nonrandomized, case-controlled comparison of surgical volumes and systematic analysis of publication volumes. Two surrogates for the proportion of thyroidectomies being performed by otolaryngologists-head and neck surgeons (OHNS) and general surgeons (GS) were chosen: 1) the operative case logs of graduates from American training programs in OHNS and GS from 1995 through 2004 were compared; and 2) the number of scientific articles published relating to thyroid surgery were systematically queried for two timeframes (1990-1994 and 2000-2004). RESULTS: There was a gradual increase in the mean number of thyroidectomies performed by GS residents from 13.2 in 1995 to 18.2 in 2004. During the same timeframe, the mean number of thyroidectomies performed by OHNS residents more than doubled from 15.0 to 33.5. The number of American GS thyroid publications from 1990 to 1994 was 79, compared with 98 in the period 2000 to 2004, representing a 24% increase. During the same timeframe, the number of American OHNS articles increased from 14 to 49 (a 250% increase). The relative proportion of thyroid publications authored by American otolaryngologists more than doubled from 15.1% to 33.3% (P = .0017). CONCLUSIONS: A clear trend is emerging in the pattern of thyroid surgery in that a growing proportion of publications are being authored by otolaryngologists compared with general surgeons, and the average number of procedures performed by graduating chief residents is now 84% higher in otolaryngology compared with general surgery.

Case-Control Studies↗

PubMed2006

Transcriptome analysis of a rat PKD model: Importance of genes involved in extracellular matrix metabolism.

Transcriptome analysis of a rat polycystic kidney disease (PKD) model: importance of genes involved in extracellular matrix metabolism. PKD is a common genetic cause of chronic renal failure, and is characterized by the accumulation of fluid-filled cysts in the kidneys and other organs. Abnormalities in the expression of selected genes thought to be involved in cystogenesis have been described, but no systematic analysis of the global transcriptomal pattern has been reported. With this aim, a rat oligomicroarray was used to identify variations in gene expression in Han:Sprague-Dawley Cy/Cy rats, an animal model presenting a severe PKD phenotype. Some upregulated genes were validated using real-time polymerase chain reaction in Cy/Cy and Cy/+ rats. Among the 350 genes identified as being upregulated, we found about 30 genes involved in extracellular matrix metabolism. These genes encoded proteins or peptides that could be implicated into two different biological processes: molecules involved in fibrosis and proteins involved in adhesion to the extracellular matrix. In heterozygotes, some genes (glypican 3, fibronectin 1) were already upregulated in early stages of the disease. We conclude that differential regulation of genes linked to extracellular matrix metabolism may be one of the first events leading to tubule enlargement and subsequent cyst formation in PKD.

Animals↗

PubMed2006

CoryneRegNet: an ontology-based data warehouse of corynebacterial transcription factors and regulatory networks.

BACKGROUND: The application of DNA microarray technology in post-genomic analysis of bacterial genome sequences has allowed the generation of huge amounts of data related to regulatory networks. This data along with literature-derived knowledge on regulation of gene expression has opened the way for genome-wide reconstruction of transcriptional regulatory networks. These large-scale reconstructions can be converted into in silico models of bacterial cells that allow a systematic analysis of network behavior in response to changing environmental conditions. DESCRIPTION: CoryneRegNet was designed to facilitate the genome-wide reconstruction of transcriptional regulatory networks of corynebacteria relevant in biotechnology and human medicine. During the import and integration process of data derived from experimental studies or literature knowledge CoryneRegNet generates links to genome annotations, to identified transcription factors and to the corresponding cis-regulatory elements. CoryneRegNet is based on a multi-layered, hierarchical and modular concept of transcriptional regulation and was implemented by using the relational database management system MySQL and an ontology-based data structure. Reconstructed regulatory networks can be visualized by using the yFiles JAVA graph library. As an application example of CoryneRegNet, we have reconstructed the global transcriptional regulation of a cellular module involved in SOS and stress response of corynebacteria. CONCLUSION: CoryneRegNet is an ontology-based data warehouse that allows a pertinent data management of regulatory interactions along with the genome-scale reconstruction of transcriptional regulatory networks. These models can further be combined with metabolic networks to build integrated models of cellular function including both metabolism and its transcriptional regulation.

Computer Graphics↗

PubMed2004

[Mechanisms of damage to metal-on-polyethylene articulating surfaces of total hip prostheses: influence of intra-articulate migration of metallic debris].

PURPOSE OF THE STUDY: The objective of this work was to identify the mechanisms leading to deterioration of the polyethylene acetabular surface in total hip prostheses. MATERIAL AND METHODS: We conducted an in vivo analysis of damage caused to articulating surfaces of metal-on-polyethylene total hip prostheses. This analysis was performed on three of the 45 explants of Harris-Galante prostheses available to use from our series of 1480 implantations performed between 1985 and 1996. These three pieces were selected because large-sized fibers of metallic debris were identified on the polyethylene surface and because the explants had been performed for reasons other than loosening in aseptic patients. Macroscopic examination was combined with scan electron microscopy to analyze morphological changes. RESULTS: We found that identical damage could be observed on the polyethylene explants despite the different sizes of the metallic heads and different metal-on-polyethylene combinations. Our analysis confirmed the deleterious effect of metallic foreign bodies trapped in contact with the articulate surfaces. These foreign bodies resulted in one case from rupture of a multifilament wire used for the trochanteric fixation and in two cases from the surfacing of the non-cemented Harris-Galante cups. The microscopic analysis demonstrated several successive localizations of the foreign bodies on the polyethylene surface before becoming deeply encrusted in the polyethylene where they deteriorated the femoral head surface (rough) and wore the polyethylene surface (abrasion). When the fibers came from the surfacing of the Harris-Galante cups, the metallic debris remained unrecognized during the revision procedure and were only identified later during the systematic examination of prosthetic explants. DISCUSSION AND CONCLUSION: These findings emphasize the importance of quality surface treatments for non-cemented prostheses and argue in favor of particular precautions during operative manipulation of these components. A systematic analysis of the prosthetic explants is advisable, particularly for components explanted because of wear or unexplained osteolysis. Use of multifilament trochanteric wires should be avoided because rupture raises the risk of intra-articulate migration of metallic debris. If the prosthesis involves a polyethylene acetabular surface, ceramic heads should be preferred to improve resistance to abrasion, particularly to limit the deleterious effect of a third body.

Adult↗

PubMed2005

"Chromatomics" the analysis of the chromatome.

Chromatin is a highly complex mixture of proteins and DNA that is involved in the regulation and coordination of gene expression within the eukaryotic nucleus. Changes in chromatin structure can convey heritable changes of gene activity in response to external stimuli without altering the primary DNA sequence. This epigenetic inheritance of particular traits very likely plays a major role during evolutionary processes. It is however, still ill-defined how this non DNA-mediated inheritance is accomplished at a molecular level. The advent of new methods to systematically study genome-wide changes in chromatin condensation, DNA methylation levels, RNA synthesis and the association of specific proteins or protein modifications now allows a thorough investigation of changes in chromatin structure and function in response to environmental alterations. We would like to review some of these global approaches and to introduce the term "chromatomics" for the systematic analysis of the DNA, RNA and protein content of the genetic material in the eukaryotic nucleus.

Chromatin↗

PubMed2001

Comparative genomic hybridization-aided unraveling of complex karyotypes in human hematopoietic neoplasias.

The information obtained by conventional cytogenetics (CC) in human leukemias is sometimes limited, in particular by complex karyotypes with many marker chromosomes. While CC is restricted to metaphases with a good quality, interphase fluorescence in situ hybridization (I-FISH) is also capable of analyzing specific anomalies in the interphase nuclei. Comparative genomic hybridization (CGH) gives additional information about the imbalanced karyotype changes in the whole genome. The aim of this study was to assess the contribution of CGH to the unraveling of complex GTG karyotypes, which are difficult to evaluate by banding analysis, and to compare these results with those by CC and FISH. Thirteen bone marrow samples and one sample obtained from peripheral blood of 13 leukemia patients were examined by CC, FISH and CGH. The GTG banding analysis showed complex karyotypes with many marker chromosomes. The most frequent abnormalities were numerical and structural aberrations on chromosomes 5 and 7. In 12 of the 14 samples, the CGH analysis was able to detect chromosomal imbalances with losses of material on chromosome 5 and 7 as the most frequent aberrations. In all 14 samples, additional FISH analyses were performed. For most of the studied neoplasias, a close correlation between CC, FISH and CGH data was observed. CGH was considerably helpful in adding additional information to classical karyotyping, if the low quality or number of metaphases was insufficient for a reliable CC analysis. Even in cases where whole chromosome painting could be applied, it added information on the breakpoints of the observed rearrangements. In only 2 of the studied 14 samples, neither CGH nor I-FISH could improve the result of karyotyping. CGH, nevertheless, can be regarded as a powerful additional technique in leukemias with unsuccessful CC, incomplete, or complex karyotypes with many marker chromosomes. A systematic analysis by three techniques such as CC, FISH and CGH guarantees an optimal genetic characterization of the neoplasias.

Acute Disease↗

PubMed2006

Delayed development and lifespan extension as features of metabolic lifestyle alteration in C. elegans under dietary restriction.

Studies of the model organism Caenorhabditis elegans have almost exclusively utilized growth on a bacterial diet. Such culturing presents a challenge to automation of experimentation and introduces bacterial metabolism as a secondary concern in drug and environmental toxicology studies. Axenic cultivation of C. elegans can avoid these problems, yet past work suggests that axenic growth is unhealthy for C. elegans. Here we employ a chemically defined liquid medium to culture C. elegans and find development slows, fecundity declines, lifespan increases, lipid and protein stores decrease, and gene expression changes relative to that on a bacterial diet. These changes do not appear to be random pathologies associated with malnutrition, as there are no developmental delays associated with starvation, such as L1 or dauer diapause. Additionally, development and reproductive period are fixed percentages of lifespan regardless of diet, suggesting that these alterations are adaptive. We propose that C. elegans can exist as a healthy animal with at least two distinct adult life histories. One life history maximizes the intrinsic rate of population increase, the other maximizes the efficiency of exploitation of the carrying capacity of the environment. Microarray analysis reveals increased transcript levels of daf-16 and downstream targets and past experiments demonstrate that DAF-16 (FOXO) acting on downstream targets can influence all of the phenotypes we see altered in maintenance medium. Thus, life history alteration in response to diet may be modulated by DAF-16. Our observations introduce a powerful system for automation of experimentation on healthy C. elegans and for systematic analysis of the profound impact of diet on animal physiology.

Animals↗

PubMedDate not supplied

Comparative analysis of the Arabidopsis and rice expressed sequence tag (EST) sets.

Large numbers of expressed sequence tags (ESTs) have now been generated from a variety of model organisms. In plants, substantial collections of ESTs are available for Arabidopsis and rice, in each case representing significant proportions of the estimated total numbers of genes. Large-scale comparisons of Arabidopsis and rice sequences are especially interesting due to the fact that these two species are representatives of the two subclasses of the flowering plants (Dicotyledonae and Monocotyledonae, respectively). Here we present the results of systematic analysis of the Arabidopsis and rice EST sets. Non-redundant sets of sequences from Arabidopsis and rice were first separately derived and then combined so that gene families in common between the two species could be identified. Our results show that 58% of non-singleton ESTs are derived from genes in gene families common to the two species. These gene families constitute the basis of a core set of higher plant genes.

Arabidopsis↗

PubMed1984

Quantitative investigation of hepatic genomic response to hormonal and pathophysiological stimuli by multivariate analysis of two-dimensional mRNA activity profiles.

We have applied techniques of multivariate analysis to the characterization and comparison of the effects of various pathophysiological and hormonal stimuli on the expression of the rat hepatic genome at a pretranslational level. In vitro translated products were resolved by two-dimensional gel electrophoresis. We analyzed 10 pathophysiological states brought about by variation in thyroidal status, starvation, administration of high carbohydrate diet, and the production of experimentally induced diabetes mellitus. Each state differed significantly from every other state in the two-dimensional electrophoretic profiles. The set consisting of the minimal number of products necessary for maintaining the distinctive patterns was identified. The analysis also defined those clusters of products that behaved in a coordinate fashion in response to the various stimuli. Lastly, the similarity and dissimilarity of hepatic mRNA activity profiles to each other could be geometrically represented in three-dimensional space. Our finding that the hepatic mRNA activity profile could distinguish reliably between closely related hormonal and pathophysiological stimuli indicates the specificity of hepatic genomic expression. A systematic analysis of such profiles may be useful as an overall index of the biologic response at the hepatocellular level.

Animals↗

PubMed2006

Gross genomic rearrangements involving deletions in the CFTR gene: characterization of six new events from a large cohort of hitherto unidentified cystic fibrosis chromosomes and meta-analysis of the underlying mechanisms.

Gross genomic rearrangements involving deletions in the CFTR gene have recently been found to account for approximately 20% of unidentified cystic fibrosis (CF) chromosomes in both French and Italian patients. Using QMPSF and walking quantitative DHPLC, six novel mutations (three simple deletions, two complex deletions with short insertions of 3-6 bp, and a complex deletion with a 182 bp inverted downstream sequence) were characterized by screening 274 unidentified CF chromosomes from 10 different countries. These lesions increase the total number of fully characterized large CFTR genomic rearrangements involving deletions to 21. Systematic analysis of the 42 associated breakpoints indicated that all 21 events were caused by nonhomologous recombination. Whole gene complexity analysis revealed a significant correlation between regions of low sequence complexity and the locations of the deletion breakpoints. Known recombination-promoting motifs were noted in the vicinity of the breakpoints. A total of 11 simple deletions were potentially explicable in terms of the classical model of replication slippage. However, the complex deletions appear to have arisen via multiple mechanisms; three of the five complex deletions with short insertions and both examples of large inverted insertions (299 and 182 bp, respectively) can be explained by either a model of serial replication slippage in cis (SRScis) or SRS in trans (SRStrans). Finally, the nature and distribution of large genomic rearrangements in the CFTR gene were compared and contrasted with those of two other genes, DMD and MSH2, with a view to gaining a broader understanding of DNA sequence context in mediating the diverse underlying mutational mechanisms.

Base Sequence↗

PubMed2001

Description of Buckarootrema goodmani n. g., n. sp. (Digenea: Pronocephalidae), a parasite of the freshwater turtle Emydura macquarii (Gray, 1830) (Pleurodira: Chelidae) from Queensland, Australia, and a phylogenetic analysis of the genera of the Pronocephalidae Looss, 1902.

Buckarootrema goodmani n. g., n. sp. is described from the small intestine of the Murray turtle, Emydura macquarii (Gray, 1830), from the vicinity of Warwick, Queensland, Australia. The distinctive taxonomic features include the vitellarium, which consists of 2 compact masses directly anterior to and occasionally overlapping the testes; the uterus with extensive pre- and postovarian coils; intestinal ceca with small, medial diverticula that terminate anterior to or at the anterior margin of the testes; a comma-shaped cirrus sac with both internal and external seminal vesicles. Phylogenetic systematic analysis of the genera of the Pronocephalidae including Buckarootrema and Notopronocephalus, the only other genus of pronocephalids reported from Australian freshwater turtles, indicates that Buckarootrema is the sister taxon of Neopronocephalus and Notopronocephalus is the sister group of the rest of the Pronocephalinae.

Animals↗

PubMed2005

Prevalence of thyroid nodules and carcinomas in patients operated on for renal hyperparathyroidism: experience with 339 consecutive patients and review of the literature.

The association between renal hyperparathyroidism (HPT) and differentiated thyroid carcinoma is discussed. To determine the prevalence and potential risk factors, we performed a retrospective analysis of our patients (1998-2004) and compared the data with the data from other surgical and autopsy studies. At our hospital, a total of 347 parathyroidectomies in 339 patients with renal HPT were performed. Most patients underwent preoperative ultrasound investigation of the thyroid gland and, if indicate, thyroid scintigraphy. Intraoperatively, both thyroid lobes were mobilized and palpated. Detected thyroid nodules were adequately resected and investigated histologically. A systematic analysis of the international literature was performed using the PubMed/MEDLINE system to identify publications on the prevalence of papillary thyroid carcinoma (PTC) in patients with renal HPT and in the overall population. Altogether, 133 patients (39.2%) underwent simultaneous thyroid surgery. The initial operation was hemithyroidectomy in 55 (16.2%), Dunhill operation in 36 (10.6%), unilateral subtotal resection in 17 (5.0%), bilateral subtotal resection in 5 (1.5%), and enucleation of a thyroid nodule in 18 (5.3%). A PTC was found in 8 of 339 patients (2.4%) and a follicular thyroid carcinoma in 1. Among 311 patients with primary cervical operation, 6 (1.9%) had a papillary thyroid carcinoma. All papillary tumors were classified as pT1 with a diameter of 1 to 12 mm; three were bifocal, and only one patient had positive lymph nodes. None of the analyzed factors showed a significant correlation with the occurrence of thyroid carcinoma. Depending on the screening method, the prevalence of occult PTC in European autopsy studies ranged from 5% to 9% and was markedly higher in almost all studies than in the present one. The prevalence of PTC in the present study makes an etiologic association between renal HPT and PTC unlikely. The clinical significance of these tumors remains unclear because all incidental tumors were small. However, if easily and safely feasible, relevant thyroid nodules should be removed during parathyroid surgery.

Adult↗

PubMed1991

Ribonuclease T1 with free recognition and catalytic site: crystal structure analysis at 1.5 A resolution.

The free form of ribonuclease T1 (RNase T1) has been crystallized at neutral pH, and the three-dimensional structure of the enzyme has been determined at 1.5 A nominal resolution. Restrained least-squares refinement yielded an R value of 14.3% for 12,623 structure amplitudes. The high resolution of the structure analysis permits a detailed description of the solvent structure around RNase T1, the reliable rotational setting of several side-chain amide and imidazole groups and the identification of seven disordered residues. Among these, the disordered and completely internal Val78 residue is noteworthy. In the RNase T1 crystal structures determined thus far it is always disordered in the absence of bound guanosine, but not in its presence. A systematic analysis of hydrogen bonding reveals the presence in RNase T1 of 40 three-center and an additional seven four-center hydrogen bonds. Three-center hydrogen bonds occur predominantly in the alpha-helix, where their minor components close 3(10)-type turns, and in beta-sheets, where their minor components connect the peptide nitrogen and carbonyl functions of the same residue. The structure of the free form is compared with complexes of RNase T1 with filled base recognition site and/or catalytic site. Several structural rearrangements occurring upon inhibitor or substrate binding are clearly apparent. In conjunction with the available biochemical knowledge, they are used to describe probable steps occurring early during RNase T1-catalyzed phosphate transesterification.

Amino Acid Sequence↗

PubMed2005

The type III-dependent Hrp pilus is required for productive interaction of Xanthomonas campestris pv. vesicatoria with pepper host plants.

The plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria expresses a type III secretion system that is necessary for both pathogenicity in susceptible hosts and the induction of the hypersensitive response in resistant plants. This specialized protein transport system is encoded by a 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Here we show that X. campestris pv. vesicatoria produces filamentous structures, the Hrp pili, at the cell surface under hrp-inducing conditions. Analysis of purified Hrp pili and immunoelectron microscopy revealed that the major component of the Hrp pilus is the HrpE protein which is encoded in the hrp gene cluster. Sequence homologues of hrpE are only found in other xanthomonads. However, hrpE is syntenic to the hrpY gene from another plant pathogen, Ralstonia solanacearum. Bioinformatic analyses suggest that all major Hrp pilus subunits from gram-negative plant pathogens may share the same structural organization, i.e., a predominant alpha-helical structure. Analysis of nonpolar mutants in hrpE demonstrated that the Hrp pilus is essential for the productive interaction of X. campestris pv. vesicatoria with pepper host plants. Furthermore, a functional Hrp pilus is required for type III-dependent protein secretion. Immunoelectron microscopy revealed that type III-secreted proteins, such as HrpF and AvrBs3, are in close contact with the Hrp pilus during and/or after their secretion. By systematic analysis of nonpolar hrp/hrc (hrp conserved) and hpa (hrp associated) mutants, we found that Hpa proteins as well as the translocon protein HrpF are dispensable for pilus assembly, while all other Hrp and Hrc proteins are required. Hence, there are no other conserved Hrp or Hrc proteins that act downstream of HrpE during type III-dependent protein translocation.

Amino Acid Sequence↗

PubMed2024

Chemical proteomics to study metabolism, a reductionist approach applied at the systems level.

Cellular metabolism encompasses a complex array of interconnected biochemical pathways that are required for cellular homeostasis. When dysregulated, metabolism underlies multiple human pathologies. At the heart of metabolic networks are enzymes that have been historically studied through a reductionist lens, and more recently, using high throughput approaches including genomics and proteomics. Merging these two divergent viewpoints are chemical proteomic technologies, including activity-based protein profiling, which combines chemical probes specific to distinct enzyme families or amino acid residues with proteomic analysis. This enables the study of metabolism at the network level with the precision of powerful biochemical approaches. Herein, we provide a primer on how chemical proteomic technologies custom-built for studying metabolism have unearthed fundamental principles in metabolic control. In parallel, these technologies have leap-frogged drug discovery through identification of novel targets and drug specificity. Collectively, chemical proteomics technologies appear to do the impossible: uniting systematic analysis with a reductionist approach.

Humans↗

PubMed1984

Synthesis, complete 1H assignments and conformations of the self-complementary hexadeoxyribonucleotide [d(CpGpApTpCpG)]2 and its fragments by high field NMR.

The two deoxyribonucleotides [d(CpGpApTpCpG)]2 and [d(CpGpCpG)]2 were synthesized by the phosphotriester method. Their duplex form under the conditions of the 1H-nmr experiments was proven by end 32P labeling with T4 polynucleotide kinase followed by butt end joining employing the absolute specificity of T4 ligase for double stranded DNA and analysis using gel electrophoresis and autoradiography. Complete nmr assignment of the 1H chemical shifts and coupling constants was achieved. The assignments were secured using sequential decoupling, NOE difference measurements, and two-dimensional COSY and SECSY experiments. Spectrum simulation confirmed the experimental values of chemical shifts and coupling constants. The techniques for the assignment outlined together with 31P and 2-D heteronuclear shift correlation permit an approach to a systematic analysis of more complex single-strand and duplex oligodeoxyribonucleotides.

Base Sequence↗