Thymidine Bases Research Articles

Etiology of craniofacial and cardiac malformations in a mouse model of SF3B4-related syndromes

Pathogenic variants in SF3B4, a component of the U2 snRNP complex important for branchpoint sequence recognition and splicing, are responsible for the acrofacial disorders Nager and Rodriguez Syndrome, also known as SF3B4-related syndromes. Patients exhibit malformations in the head, face, limbs, vertebrae as well as the heart. To uncover the etiology of craniofacial malformations found in SF3B4-related syndromes, mutant mouse lines with homozygous deletion of Sf3b4 in neural crest cells (NCC) were generated. Like in human patients, these embryos had craniofacial and cardiac malformations with variable expressivity and penetrance. The severity and survival of Sf3b4 NCC mutants was modified by the level of Sf3b4 in neighboring non-NCC. RNA sequencing analysis of heads of embryos prior to morphological abnormalities revealed significant changes in expression of genes forming the NCC regulatory network, as well as an increase in exon skipping. Additionally, several key histone modifiers involved in craniofacial and cardiac development showed increased exon skipping. Increased exon skipping was also associated with use of a more proximal branch point, as well as an enrichment in thymidine bases in the 50 bp around the branch points. We propose that decrease in Sf3b4 causes changes in the expression and splicing of transcripts required for proper craniofacial and cardiac development, leading to abnormalities.

Behind Base J: The Roles of JBP1 and JBP2 on Trypanosomatids

β-D-glucopyranosyloxymethiluracil (Base J) is a modified thymidine base found in kinetoplastids and some related organisms. Interestingly, Base J distribution into the genome can vary depending on the organism and its life stage. Base J is reported to be found mostly at telomeric repeats, on inactive variant surface glycoproteins (VSG's) expression sites (e.g., T. brucei), in RNA polymerase II termination sites and sub-telomeric regions (e.g., Leishmania). This hypermodified nucleotide is synthesized in two steps with the participation of two distinct thymidine hydroxylases, J-binding protein 1 and 2 (JBP1 and JBP2, respectively) and a β-glucosyl transferase. A third J-binding protein, named JBP3, was recently identified as part of a multimeric complex. Although its structural similarities with JBP1, it seems not to be involved in J biosynthesis but to play roles in gene expression regulation in trypanosomatids. Over the years, with the characterization of JBP1 and JBP2 mutant lines, Base J functions have been targeted and shone a light on that matter, showing genus-specific features. This review aims to explore Base J's reported participation as a regulator of RNA polymerase II transcription termination and to summarize the functional and structural characteristics and similarities of the remarkable JBP proteins in pathogenic trypanosomatids.

Probing ATP/ATP-Aptamer or ATP-Aptamer Mutant Complexes by Microscale Thermophoresis and Molecular Dynamics Simulations: Discovery of an ATP-Aptamer Sequence of Superior Binding Properties.

Microscale thermophoresis (MST) is used to follow the dissociation constants corresponding to ATTO 488-labeled adenosine triphosphate (ATP) and the ATP-aptamer or ATP-aptamer mutants that include two binding sites for the ATP ligand. A set of eight ATP-aptamer mutants, where the thymidine bases, within the reported ATP binding aptamer sites, are substituted with cytosine bases, are examined. The MST-derived dissociation constant of ATP to the reported aptamer is Kd = 31 ± 3 μM, whereas most of the aptamer mutants show lower affinity (higher Kd values) toward the ATP ligand. One aptamer mutant reveals, however, a higher affinity toward the ATP ligand, as compared to the reported ATP-aptamer. Molecular dynamics and docking simulations identify the structural features that control the affinities of binding of the ATP ligand to the two binding sites associated with the ATP-aptamer or the ATP-aptamer mutants. The simulated structures suggest that H-bonds between the ATP ligand and G9 and G11 bases, within one binding domain, and the π-π interactions between G6 and the ATP purine moiety and the pyrimidine ring, in the second binding domain, control the affinity of binding interactions between the ATP ligand and the ATP-aptamer or ATP-aptamer mutant. Very good correlation between the computed Kd values and the MST-derived Kd values is found. The ATP-aptamer mutant (consisting ofA1→ G,T4 → C, T12 → C, A24→ G,and T27 → C mutations) reveals superior binding affinities toward the ATP ligands ( Kd = 15 ± 1 μM) as compared to the binding affinity of ATP to the reported aptamer. These features of the mutant are supported by molecular dynamics simulations.

Discovery and functional implications of a miR-29b-1/miR-29a cluster polymorphism in acute myeloid leukemia.

We previously reported that microRNA (miR)-29b is down-regulated and has a tumor suppressor role in acute myeloid leukemia (AML). However, little is known about the mechanisms responsible for miR-29b expression downregulation in AML. In this work we screened for mutations that could affect miR-29b expression. Using Sanger sequencing, we identified a germline thymidine (T) base deletion within the miR-29b-1/miR-29a cluster precursor in 16% of AML patients. Remarkably we found a significant enrichment for the presence of the miR-29 polymorphism in core binding factor (CBF) newly diagnosed AML patients (n = 61/303; 20%) with respect to age, sex and race matched controls (n = 43/402:11%, P < 0.01). Mechanistically, this polymorphism affects the expression ratio of mature miR-29b and miR-29a by dampening the processing of miR-29a. RNA immunoprecipitation assays showed reduced DROSHA binding capacity to the polymorphism with respect to the controls. Finally, we showed that this polymorphism negatively impacts the ability of miR-29b-1/miR-29a cluster to target MCL-1 and CDK6, both known miR-29 targets.

Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision

TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034–encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5′ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).

Detection of beer spoilage bacteria Pectinatus and Megasphaera with acridinium ester labelled DNA probes using a hybridisation protection assay

DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of the genera Pectinatus and Megasphaera using a hybridisation protection assay (HPA). All the probes were modified during synthesis by addition of an amino linker arm at the 5′ end or were internally modified by inserting an amine modified thymidine base. Synthesised probes then were labelled with acridinium ester (AE) and purified using reverse phase HPLC. The internally AE labelled probes were able to detect target RNA within the range of 0.016–0.0032pmol. All the designed probes showed high specificity towards target RNA and could detect bacterial contamination within the range of ca. 5×1021×103 CFU using the HPA. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media, routinely used in brewing laboratories.

Electronic transport through short dsDNA measured with mechanically controlled break junctions: New thiol–gold binding protocol improves conductance

It is shown that double‐stranded DNA which is directly coupled to gold via a modified thymidine base exhibits a higher conductance than reported for DNA coupled to metal electrodes using different binding schemes. The measurements of electrical conductance are performed in a mechanically controlled break junction setup in aqueous solution and in high vacuum at room temperature. The current–voltage characteristics obtained in vacuum can be understood if a single molecular energy level determines the transport.

9239 POSTER Rapid Method to Measure Thioguanine Incorporation Into DNA

Background: The thiopurine drugs, 6-mercaptopurine, azathioprine, and thioguanine, are used in the treatment of acute lymphoblastic leukaemia (ALL). During treatment the thioguanine nucleotides formed are incorporated into the DNA, causing apoptosis due to the cells inability to repair the resulting damage. This mechanism is believed to be important for the effects of thiopurine drugs. We have developed a novel method for the determination of thioguanine incorporation into DNA which is both faster and cheaper than earlier methods.Monitoring the effects of thiopurine treatment by measuring thiopurine metabolites in erythrocytes has proven to be elusive due to the lack of good correlation between measured concentrations and thiopurine effects. If the incorporation is the main mechanism of thiopurine action, a reliable method capable of measuring the incorporation in an ordinary blood sample, such as the method we have developed, should provide a significantly better correlation with treatment effect.Material and Methods: Briefly, DNA extracted from buffy coat is degraded using nuclease P1 and alkaline phosphatase to produce free nucleosides which are purified by filtration. Thioguanosine and thymidine are separated and detected using an LC-MS/MS system and the ratio between the bases provides a measurement of the extent of thioguanine incorporation in DNA. The method has been successfully applied to cell culture samples as well as samples from patients treated orally with thiopurines.Results: In 8 inflammatory bowel disease patients treated with azathioprine the measured incorporation ranged from 2.2 to 8.4 thioguanine bases for every 10 000 thymidine bases (median 5.2). This is in agreement with earlier reports on incorporation in childhood leukemia patients.Conclusions: With the presented method it is possible to determine the incorporation of thioguanine into DNA during thiopurine treatment in a cost effective manner, but further research is needed to determine if there is a place for this type of methods in the monitoring of thiopurine treatment.An ongoing study aims to compare the incorporation to treatment effects as well as conventional measurements of erythrocyte metabolite levels. By this study we hope to determine if incorporation is a more reliable measurement to predict treatment effect and if the erythrocyte metabolite levels correlate with the incorporation.

New familial heterozygous c 4066_4068 delTT 2 bp deletion of the SCN5A gene causing Brugada syndrome

Cardiac sodium channel alterations have been identified as the underlying condition in patients with Brugada syndrome. This study identified a novel mutation of the SCN5A gene in a family with Brugada syndrome. Blood was drawn from the children and mother for genetic analysis. All exons of the SCN5A gene were amplified by polymerase chain reaction, and a sequence analysis was performed. The mutation was detected in 1 symptomatic and 2 asymptomatic family members. The deletion of base 4066_4068delTT leads to a shift in the amino acid sequence and a premature stop of the protein translation. The clinical diagnosis of Brugada syndrome in this family was supported by the detection of the new mutation. We describe a family partly with Brugada syndrome and a novel mutation in the exon 23 of the SCN5A gene leading to a deletion of 2 thymidine bases. This mutation results in an early termination of the encoded protein and possibly in a nonfunctional channel protein.

Discovery and Functional Implications of a Novel Mir-29b/Mir-29a Polymorphism in Acute Myeloid Leukemia.

Abstract 1975Poster Board I-998 Background:MicroRNAs (miRNAs) are associated with cytogenetics and molecular subtypes of acute myelogeneous leukemia (AML). We have previously shown that miR-29 expression is down-regulated in cytogenetically normal AML (CN-AML) with wild type NPM1 and in t(11q23) primary blasts. Functionally, restoration of miR-29b in AML cell lines and primary samples induces apoptosis and dramatically reduces tumorigenicity in a xenograft leukemia model (Garzon et al, EHA 2008). Despite, these studies supporting a tumor suppressor role of miR-29b in AML, little is known about how miR-29 expression is down-regulated in AML. Since, miRNAs could be target for mutation, here we propose to screen mutations that could affect miR-29 expression and function. Methods:The miR-29 family is comprised of three isoforms arranged in 2 clusters; miR-29b-1 and -a located on chromosome 7q32 and miR-29b-2 and -c located on chromosome 1q23. To screen for mutations, the entire genomic region from blasts of 100 primary AML samples corresponding to the miR-29b-1 and -a cluster, including 200 bp at the 5' and 3' ends was amplified and sequenced using the Applied Biosystems DNA sequencing system. When a deviation from the normal sequence was found, a panel of DNA from the blood of 50 control subjects was screened to identify polymorphisms. Patient characteristics include: CN-AML: 62 (FLT3-ITD 10/43, NPM1 mutated (34/62); inv16: 10; t(8;21): 2; t11q23: 2; complex karyotype (CK): 10; monosomy 7(-7): 7; other cytogenetics: 7. miR-29 expression we performed by miRNA Taqman assays as per manufacturer recommendations. Results:We identified a thymidine (T) base deletion within the miR-29b-1 and -a cluster precursor miRNAs (at -264 bp from the 5' position of miR-29a in chromosome 7q32) in 17/100 patients. The (T) base deletion was observed in 4/10 inv16 and 6/62 CN-AML patients, while the other 7 cases were distributed among CK (2/10), -7 (3/7), 11q23 (1/2) and other cytogenetics (1/7). In 2 patients, normal cells from the buccal mucosa were heterozygous for this abnormality. The frequency of this germline abnormality in the normal population was 16% (8/50 cases). Next, we investigated the miR-29b and -a expression in 35/100 primary AML samples, where RNA was also available. Although miR-29a and -b levels were not significantly different in polymorphism (n=10) versus wild type (WT) (n=35) samples, we observed that the miR-29a/miR-29b ratios were significantly lower in the polymorphism than WT (43.5 vs. 24.9 respectively, P-value=0.007, t-test). To characterize further this abnormality, we cloned the polymorphism harboring miR-29b and -a cluster from 1 patient into p-Retro Super plasmid and transfected into K562 cells (lack miR-29 expression) along with WT and empty vector constructs. Northern blotting after 24-48 hours revealed an accumulation of the precursor miR-29a while the mature miR-29a level was decreased by 2 fold. The level of the mature miR-29b was unchanged. To asses whether this polymorphism affects miR-29 targeting efficiency, we co-transfected a reporter luciferase construct containing the 3' untranslated region of the known miR-29 target, MCL-1 with the WT, empty vector and polymorphism harboring miR-29b and -a cluster and performed luciferase assays. Interestingly, relative normalized luciferase activities were less inhibited with the polymorphism cluster than the WT construct (relative reduction WT:80%, Polym:63%. Likewise, MCL-1 protein down-regulation elicited by the ectopic WT cluster overexpression was stronger than the one observed for the polymorphism harboring cluster (b-actin/MCL-1 rations 0.35 vs 0.48, respectively). Conclusions:Our results identify a novel germline polymorphism within the miR-29b and —a cluster in AML. The frequency of this polymorphism in AML is similar to the normal population. However, the increased frequency observed in the inv16 subgroup (4/10) warrant further confirmation in a large cohort of patients. Functionally, this polymorphism affects the expression ratio of miR-29b and —a by dampening the processing of miR-29a and impacts negatively in the ability of this cluster to target the oncogene MCL-1. Disclosures:No relevant conflicts of interest to declare.

Synthesis and Characterization of DNA Containing anN1-2′-Deoxyinosine-ethyl-N3-thymidine Interstrand Cross-Link: A Structural Mimic of the Cross-Link Formed by 1,3-Bis-(2-chloroethyl)-1-nitrosourea

Interstrand cross-links, which are generated by chemotherapeutic treatment with bis-alkylating agents, exert their therapeutic effect by connecting the nucleobases of adjacent DNA strands together and represent some of the most threatening forms of damage suffered by genomic DNA. However, one of the reasons for treatment failure using these agents is due to enhanced repair of this DNA damage. The pursuit of understanding the repair of interstrand cross-links by repair systems has necessitated the synthesis of sufficient quantities of such damaged DNA. We report the synthesis of a site-specific interstrand cross-linked duplex containing an ethylene-bridged N (1)-2'-deoxyinosine- N (3)-thymidine base pair prepared by solution and solid-phase synthesis as a mimic for the lesion formed by the therapeutic agent 1,3-bis-(2-chloroethyl)-1-nitrosourea using both a phosphoramidite and a bis-phosphoramidite approach. UV thermal denaturation experiments revealed that this cross-linked duplex was stabilized by 52 degrees C relative to the noncross-linked control, and circular dichroism studies indicated little deviation from a B-form structure compared to a duplex that contained a G-C base pair at the same position. Molecular models of the cross-linked duplex that were geometry optimized using the AMBER forcefield also suggest that this lesion induces minimal distortion in B-form DNA. This modified oligonucleotide will be useful for studies related to the investigation of interstrand cross-linked DNA repair.

XRCC1 down‐regulation in human cells leads to DNA‐damaging agent hypersensitivity, elevated sister chromatid exchange, and reduced survival of BRCA2 mutant cells

Previous studies using rodent cells indicate that a deficiency in XRCC1 results in reduced single-strand break repair, increased sensitivity to DNA-damaging agents, and elevated levels of sister chromatid exchange (SCE). Epidemiological studies have suggested an association of certain human XRCC1 polymorphisms with genetic instability and cancer susceptibility. However, investigations on the molecular functions of XRCC1 in human cells are limited. To determine the contributions of this nonenzymatic scaffold protein, we suppressed XRCC1 levels in several human cell lines using small interfering RNA (siRNA) technology. We report that XRCC1 down-regulation in HeLa cells leads to a concomitant decrease in the DNA ligase 3 protein level and an impaired nick ligation capacity. In addition, depletion of XRCC1 resulted in a significantly increased sensitivity to the alkylating agent methyl methanesulfonate and the thymidine base analog 5-hydroxymethyl-2'-deoxyuridine, a slightly increased sensitivity to ethyl methanesulfonate and 1,3-bis(2-chloroethyl)-1-nitrosourea, and no change in the response to camptothecin. We also discovered that a 70-80% reduction in XRCC1 protein leads to an elevated level of SCE in both HeLa cells and normal human fibroblasts, but does not affect chromosome aberrations in the diploid fibroblasts. Last, XRCC1 siRNA transfection led to an approximately 40% decrease in the survival of BRCA2-deficient cells, supporting a model whereby the accumulation of unrepaired SSBs leads to the accumulation of cytotoxic DNA double strand breaks following replication fork collapse in cells defective in homologous recombination.

Mispair-aligned N3T-alkyl-N3T interstrand cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of variable lengths.

Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized a cross-link that bridges the N(3) positions of a mismatched thymidine base pair. This cross-link, which is similar to the N(3)C-alkyl-N(3)C cross-link that has been observed between mismatched cytosine base pairs, was introduced by first incorporating a cross-linked phosphoramidite unit at the 5'-end of an oligonucleotide chain. Fully cross-linked duplexes were then synthesized using an orthogonal approach to selectively remove protecting groups, thus allowing construction of the cross-linked duplex via conventional solid-phase oligonucleotide synthesis. Short DNA duplexes with alkyl cross-links of various lengths (two, four, and seven methylene units) were prepared, and their physical properties were studied via UV thermal denaturation and circular dichroism spectroscopy. These linkers were found to stabilize the duplexes by 37, 31, and 16 degrees C for the two-, four-, and seven-carbon linkers, respectively, relative to a non-cross-linked duplex. Circular dichroism spectra suggested that these lesions induce very little deviation in the global structure relative to the non-cross-linked duplex DNA control. Molecular models show that the two-carbon cross-link spans the distance between the N(3) atoms of the T-T mismatch without perturbing the helix structure, whereas the longer linkers, particularly the seven-carbon linker, tend to push the thymines apart, creating a local distortion. This perturbation may account for the lower thermal stability of the seven-carbon versus two-carbon cross-linked duplex.

19F NMR Studies of Vaccinia Type IB Topoisomerase: CONFORMATIONAL DYNAMICS OF THE BOUND DNA SUBSTRATE

The site-specific DNA cleavage and religation activities of the vaccinia virus type IB topoisomerase at (C/T)CCTT(+1)X(-1) sites in duplex DNA have allowed detailed investigations of the chemical and conformational steps on the reaction pathway of this enzyme (see accompanying article (Kwon, K., and Stivers, J. T. (2002) J. Biol. Chem. 277, 345-352)). To extend these studies to the DNA substrate, we have performed 19F NMR experiments using substrates in which the +1 T has been replaced with the NMR-sensitive thymidine base analogue 5-fluoro-2'-deoxyuridine (5-F-dUrd). Substitution of 5-F-dUrd has little effect on the binding affinity of topoisomerase I for DNA, results in small changes in the cleavage and religation rate constants, and produces a net 3-fold decrease in the cleavage equilibrium constant as compared with the CCCTT consensus DNA. One-dimensional 19F NMR experiments show that the +1 5-F-dUrd is in a dynamic equilibrium between a stacked and unstacked state in both the noncovalent complex and the covalent phosphotyrosine complex. These NMR observations are supported by the selective sensitivity of the +1 T and +1 5-F-dUrd to KMnO4 oxidation. A role for localized DNA distortion in the topoisomerase I mechanism is suggested.

From equilibrium to kinetic footprinting of RNA structure

RNA is a central molecule in the transmission, processing, and translation of genetic information. Although the critical role of RNA in cellular processes has long been recognized, the discoveries of autocatalytic 1 and catalytic processes 2,3 that are RNA-based have spurred even more interest in the chemistry and biology of RNA systems. The unique three dimensional structures adopted by various RNAs determine their activity and thus an understanding of structure is key to a full appreciation of their biological roles. Advances in high-resolution structure determination have provided insight into the organization and function of increasingly complex biochemical entities involved in RNA processing culminating in the recent structure of the large ribosomal subunit. Despite these impressive achievements, structural analysis by X-ray or NMR techniques remains an arduous and sometimes capricious endeavour. There is thus a need for simple chemical probes to provide structural information in the absence of higher resolution data and to complement those data where they have been obtained. Chemical tools for the analysis of nucleic acid structure were largely developed for use in characterization of DNA or protein–DNA complexes. Because RNA differs from DNA only by the presence of a 2 -hydroxy group, methodologies developed for the study of DNA systems have proven useful in the study of RNA structure and function as well. Many of these tools are reagents which react with some nucleic acid functionality in a specific fashion and render the phosphodiester backbone labile to cleavage. RNAs of different sizes, radio-labeled at the 5 or 3 ends with P, may be separated at nucleotide resolution by denaturing polyacrylamide gel electrophoresis (PAGE) and then visualized by exposure of the gel to either X-ray film or another imaging surface. Nucleic acid fragments appear as bands in the gel; smaller fragments migrate faster than larger ones and the exact position in a particular sequence may be ascertained by comparison to a “ladder” of bands generated by partial alkaline hydrolysis, base-specific chemical sequencing, enzymatic sequencing, or base-specific enzymatic cleavage (the enzyme RNaseT1, for example, cleaves RNA sequences only after guanosine residues). When a chemical reagent is used to induce cleavage of the RNA backbone, the presence of a band on a gel indicates that the targeted functionality was accessible to the modifying reagent while the absence of a band indicates that the functionality was inaccessible due to the formation of some kind of interor intra-molecular structure. Regions of decreased band intensity spanning one or more nucleotides are referred to as footprints and this type of experiment, known as protection footprinting, has been of immense power in the analysis of structure in nucleic acid systems (Fig. 1; interference footprinting involves pre-treatment of a nucleic acid with a modifying reagent in order to determine which modifications block structure formation). A large group of reagents have been used in protection footprinting experiments. Gilbert and co-workers showed that dimethyl sulfate (DMS), which reacts with N7 of guanosine and N3 of adenosine, can be used to detect contacts to these positions. Diethyl pyrocarbonate† modification of DNA has been used as a probe of contacts to the N7 positions of G and A while osmium tetraoxide (OsO4) and potassium permanganate (KMnO4), which react with the 5,6 bond of the thymidine base, have been used as protection probes of changes in DNA helix parameters. Protection footprinting has typically been used to compare two different states in a system; for example, an unbound nucleic acid can be compared to a protein-bound nucleic acid or an unstructured nucleic acid can be compared to a structured or folded nucleic acid (Fig. 1). There have been a host of protection footprinting studies of protein–DNA systems ranging Fig. 1 Protection footprinting as a probe of nucleic acid structure. a) Formation of higher order structure in nucleic acids by protein–nucleic acid association (top) or folding of a nucleic acid (bottom). The structure of the complexed or folded molecule may be probed by treatment with a nucleic acid modifying agent which initiates strand cleavage. Arrows represent modification of the nucleic acid with such a reagent and shading denotes protection from modification; b) PAGE analysis of P radio-labeled fragments obtained in the protection experiment. Comparison is made of the cleavage patterns obtained for the unbound or unstructured state (A) with the bound or structured state (B). Areas of reduced band intensity are referred to as footprints and represent the protection from chemical modification resulting from protein binding or higher order structure formation.

Comparative Stability of Triple Helices Containing Modified DNA or RNA Pyrimidine Strands

Control of gene expression by oligodeoxyribonucleotide-directed triplex formation, known as antigene therapy (l), is widely studied since works have demonstrated the sequence-specific recognition of double helical DNA by a third strand (2,3). Triplex formation occurs when an oligopyrimidine strand binds in the major groove with a parallel orientation to the purine strand of polypurine polypyrimidine DNA duplex. Binding specificity is obtained from recognition of thymidine and AT base pair and N3 protonated cytosine and GC base pairs to form respectively the isomorphous T:AT and C+:GC triplets via Hoogsteen hydrogen bonding (2,3). Tfiple helix formation involving cytosine is pH dependent. In order to use oligos for antigene therapy, they must fulfill at least two requirements such as nucleases resistance, since wild type DNA and RNA are rapidly degraded by nucleases present in cells and sera (4), and high binding affinity with the complementary double-stranded DNA in order to block the transcription or the replication of the corresponding gene. To accomplish the former point, several modifications of the phcc;We or of the sugar have been proposed (5).

Chemical Cleavage of Heteroduplex DNA to Identify Mutations

Mutation screening by the chemical-cleavage method is based on the fact that mismatched cytosine (C) and thymidine (T) are more reactive with the compounds hydroxylamine and osmium tetroxide, respectively, than are Watson and Crick-paired cytosine and thymidine bases. In this protocol, an excess of unlabeled target DNA is hybridized with labeled wild-type DNA probe and heteroduplexes are formed. One aliquot is treated with hydroxylamine, which reacts with mismatched C bases. Another aliquot is treated with osmium tetroxide, which reacts with mismatched T bases. The reactions are mixed with piperidine; the strands are then cleaved at the sites where hydroxylamine and osmium tetroxide react. Cleaved fragments are then electrophoresed and sized on polyacrylamide gels, identifying the point of cleavage (and hence the position of the mutation). Then only a small portion of the mutant gene needs to be sequenced to define a single change between two DNA sequences.

Dynamics in psoralen-damaged DNA by 1H-detected natural abundance 13C NMR spectroscopy.

The dynamics of the DNA oligomer d(GCGTACGC)2 and the 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen-DNA furanside monoadduct (MAf) of this oligomer have been determined from NMR relaxation parameters. Longitudinal and transverse 13C relaxation rates and heteronuclear NOE relaxation data have been measured at natural abundance and have been analyzed in the context of the Lipari and Szabo model-free formalism. The generalized order parameters for methine carbons in the octamer sequence d(GCGTACGC)2 (UM) are relatively and uniformly high for the entire molecule. The generalized order parameters for methine carbons in the MAf are significantly lower for the deoxyribose bearing the damaged thymidine base and for the bases flanking the lesion on the undamaged strand, indicating additional conformational flexibility due to the lesion. The order parameters for the bases on the damaged strand flanking the lesion remain high. Analysis of the relaxation data indicates substantial chemical exchange for the adenosine residues in the UM TpA site, and this chemical exchange is quenched upon MAf formation. These data are discussed in terms of a model for DNA damage recognition by the nucleotide excision repair system.

A sequence variation: 713-8delC in the transforming growth factor-beta 1 gene has higher prevalence in osteoporotic women than in normal women and is associated with very low bone mass in osteoporotic women and increased bone turnover in both osteoporotic and normal women

Bone mass is partly genetically determined. The genes involved are, however, still largely unknown. Transforming growth factor-beta 1 (TGF-β1) is considered a putative regulator of osteoclastic-osteoblastic interaction (coupling). The aim of the present study was therefore to examine whether possible variants of the TGF-β1 gene are related to bone mass and osteoporosis. We examined 161 osteoporotic women (at least one low energy spinal fracture) and 131 normal women. We investigated sequence variations in the TGF-β1 gene using the single-stranded conformation polymorphism (SSCP) technique combined with DNA sequencing. Seven patients were heterozygous for a cytosine to thymidine base substitution at position 76 in exon 5 (C 788-T) (corresponding to position 788 in the TGF-β1 cDNA), resulting in a threonine to isoleucine amino acid shift at position 263 in the TGF-β1 propeptide (Thr 263-Ile). Ten other patients had a one base deletion in the intron sequence 8 bases prior to exon 5 (713-8delC), which could influence splicing. Five normal women exhibited the C 788-T sequence variant, and two the 713-8delC. The prevalence of 713-8delC was significantly higher in the osteoporotic group ( χ 2 = 4.02, p < 0.05). Osteoporotic patients with the 713-8delC variant had increased levels of bone alkaline phosphatase ( p < 0.05). If the osteoporotic patients with a z score of the lumbar spine below −1 were examined separately, we found increased serum levels of bone alkaline phosphatase ( p < 0.05), increased urinary excretion of hydroxyproline ( p < 0.05), and reduced bone mass of the lumbar spine ( p < 0.05),in patients with 713-8delC. No correlation to bone mass was demonstrated in the normal women, but 713-8delC was associated with increased serum levels of bone alkaline phosphatase ( p < 0.05). The sequence variation, 713-8delC, in the TGF-β1 gene is more frequent in patients with osteoporosis compared to normal controls. The 713-8delC variant seems to be associated with very low bone mass in osteoporotic women with low bone mass and increased bone turnover in both osteoporotic and normal women.

Fast capillary electrophoresis-laser induced fluorescence analysis of ligase chain reaction products: human mitochondrial DNA point mutations causing Leber's hereditary optic neuropathy.

High speed capillary electrophoresis-laser-induced fluorescence (CE-LIF) has been used to separate and detect point mutations using the ligase chain reaction (LCR). The method utilizes short capillary columns (7.5 cm effective length) and fields of 400 V/cm to analyze DNA-ethidium bromide complexes using an He/Ne laser. The method was first demonstrated with a commercially available kit for LCR based on a lacI gene fragment inserted in a Bluescript II phagemid. LCR-CE-LIF was then applied to detect point mutations in human mitochondrial DNA, resulting in Leber's hereditary optic neuropathy (LHON). Three severe mutations were analyzed in which the original base is substituted by a thymidine base at positions 3460, 11778 and 14459. Appropriate primers were designed with polyT tails for length discrimination of pooled samples. Successful detection of mutated samples was achieved, with appropriate correction for small amounts of nonspecific ligated product. The method is rapid, easy to implement, and automatable.