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Results for “duplex sequencing”

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PubMed2004

A library of siRNA duplexes targeting the phosphoinositide 3-kinase pathway: determinants of gene silencing for use in cell-based screens.

Gene silencing through RNA interference (RNAi) has been established as a means of conducting reverse genetic studies. In order to better understand the determinants of short interfering RNA (siRNA) knockdown for use in high-throughput cell-based screens, 148 siRNA duplexes targeting 30 genes within the PI3K pathway were selected and synthesized. The extent of RNA knockdown was measured for 22 genes by quantitative real-time PCR. Analysis of the parameters correlating with effective knockdown showed that (i) duplexes targeting the middle of the coding sequence silenced significantly poorer, (ii) silencing by duplexes targeting the 3'UTR was comparable with duplexes targeting the coding sequence, (iii) pooling of four or five duplexes per gene was remarkably efficient in knocking down gene expression and (iv) among duplexes that achieved a >70% knockdown of the mRNA there were strong nucleotide preferences at specific positions, most notably positions 11 (G or C) and 19 (T) of the siRNA duplex. Finally, in a proof-of-principle pathway-wide cell-based genetic screen, conducted to detect negative genetic regulators of Akt S473 phosphorylation, both known negative regulators of this phosphorylation, PTEN and PDK1, were found. These data help to lay the foundation for genome-wide siRNA screens in mammalian cells.

Cell Line↗

PubMed2004

Multiparametric duplex real-time nucleic acid sequence-based amplification assay for mRNA profiling.

Nucleic acid sequence-based amplification (NASBA) is a sensitive isothermal transcription-based amplification method. We have developed real-time NASBA assays to detect mRNA coding for the estrogen receptor alpha (ESR1) and the progesterone receptor (PGR) in breast tumors by means of duplex reactions using cyclophilin B (PPIB) as the normalizing gene. Both the ESR1/PPIB and PGR/PPIB duplex NASBA assays are highly sensitive, specific, and reproducible. Quantification is determined using external standard calibration curves and the ratio between the number of target and housekeeping gene mRNA copies. Amplification of the target gene in the duplex NASBA assay was disrupted when this latter was mixed with a large amount of the housekeeping PPIB gene, suggesting that it is preferable for the normalizing gene chosen to have an expression level comparable to the target gene. Sensitivity and robustness of the duplex NASBA assays were assessed in breast cancer cell lines. Such a rapid and easy-to-use multiparametric duplex real-time NASBA assay could also advantageously be set up for other mRNA profiling applications.

Biological Assay↗

PubMed1987

Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis.

We have used a new method of DNA analysis for the rapid prenatal diagnosis of sickle cell anemia in two fetuses at risk for this disease. This method of detecting the sickle gene is a modification of standard restriction-enzyme techniques and requires only a small amount of DNA. The first step involves a 200,000-fold enzymatic amplification of the specific beta-globin DNA sequences that may carry the sickle mutation. This provides a sufficient quantity of DNA for the analysis. Next, a short radiolabeled synthetic DNA sequence homologous to normal beta A-globin gene sequences is hybridized to the amplified target sequences. The hybrid "duplexes" are then digested sequentially with two restriction endonucleases. The presence of beta A- or beta S-globin gene sequences in the amplified target DNA from the patient determines whether the beta A-hybridization probe anneals perfectly or with a single nucleotide mismatch. This difference affects the restriction-enzyme digestion of the DNA and the size of the resulting radiolabeled digestion products, which can be distinguished by electrophoresis followed by autoradiography. This method is sufficiently sensitive and rapid that the prenatal diagnosis of sickle cell anemia can be made on the same day that the fetal DNA is made available. It can also be applied to the diagnosis of hemoglobin C disease.

Anemia, Sickle Cell↗

PubMed1990

Circular dichroism studies of an oligodeoxyribonucleotide containing a hairpin loop made of a hexaethylene glycol chain: conformation and stability.

An oligodeoxyribonucleotide, d(GCTCACAAT-X-ATTGTGAGC), where X represents a hexaethylene glycol chain, was studied using circular dichroism spectroscopy. Its conformation and conformational stability were compared to those of compounds where X was replaced by four thymines and to the duplex of same sequence without loop. The compound with the hexaethylene glycol chain can form a hairpin looped structure as well as a bulged duplex structure. In both cases the duplex region of the oligodeoxyribonucleotide exhibits the same conformation. In similar conditions the oligodeoxyribonucleotide with a four thymines loop forms exclusively a hairpin structure. Comparison between the thermodynamic parameters (delta H, delta S, delta G) associated with the formation of the structure of the three compounds are presented. In the case of the compound with the hexaethylene glycol chain it is shown that the large increase in its melting temperature (by about 35 degrees C in our experimental conditions) when compared to the non looped structure is mainly due to the fact that its melting process is intramolecular (monomolecular) whereas the other one is bimolecular.

Base Sequence↗

PubMed1995

Crystal structure of a DNA decamer showing a novel pseudo four-way helix-helix junction.

The crystal structure of the decanucleotide d(CGCAATTGCG)2 has been solved by a combination of molecular replacement and heavy-atom procedures and has been refined to an R factor of 20.2% at 2.7 A. It is not a fully base-paired duplex but has a central core of eight Watson-Crick base pairs flanked by unpaired terminal guanosines and cytosines. These participate in hydrogen-bonding arrangements with adjacent decamer duplexes in the crystal lattice. The unpaired guanosines are bound in the G+C regions of duplex minor grooves. The cytosines have relatively high mobility, even though they are constrained to be in one region where they are involved in base-paired triplets with G.C base pairs. The 5'-AATT sequence in the duplex region has a narrow minor groove, providing further confirmation of the sequence-dependent nature of groove width.

Base Composition↗

PubMed1977

Rauscher-leukemia-virus-related sequences in human DNA: presence in some tissues of some patients with hemotopoietic neoplasias and absence in DNA from other tissues.

A [3H[cDNA probe synthesized from the RNA genome of Rauscher murine leukemia virus (MuLVR) and purified by hybridization to MuLVR70S RNA was hybridized to DNA from human normal and hemotopoietic neoplasia tissues. This cDNA hybridized completely to its homologous 70S RNA and was free of self-complementary sequences. Sequences complementary to MuLVR cDNA were found in DNA from tissues of some patients with leukemia (2 of 8), Hodgkin's disease (3 of 10), and one patient with multiple myeloma. DNA from spleen and kidney of a patient with nonneoplastic disease did not contain detectable MuLVR-related sequences. These virus-related sequences in the DNA from these neoplastic tissues were related but not identical to MuLVR sequences because differences of approximately 6 degrees in the midpoints of thermal elution profiles were found between the heterologous and homologous duplexes. These nucleotide sequences are not the same as the proviral sequences of baboon type-C virus previously found from some other patients with leukemia [Reitz et al. (1976) Proc. Natl. Acad. Sci. USA 73,2113-2117; Wong-Staal et al. (1976) Nature 262, 190-195], because there is no sequence homology between nucleic acids from MuLVR and baboon virus. The absence of these nucleic acid sequences in many tissues of patients with neoplasia and from the few tissues examined from people with nonneoplastic disease suggests that they are not endogenous elements but are acquired after fertilization. Taken together with the previous detection of baboon and woolly monkey type-C viral related components in some human tumors, the results suggest acquisition of at least three types of type-C viral sequences in the human population.

Base Sequence↗

PubMed2003

A fluorescence-based high-throughput screening assay for inhibitors of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H activity.

A fluorescence resonance energy transfer assay readily applicable to 96-well and 384-well microplate formats with robotic operation was developed to enable high-throughput screening for inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT)-associated RNase H activity, an underexplored target for antiretroviral development. The assay substrate is an 18-nucleotide 3'-fluorescein-labeled RNA annealed to a complementary 18-nucleotide 5'-Dabcyl-modified DNA. The intact duplex has an extremely low background fluorescent signal and provides up to 50-fold fluorescent signal enhancement following hydrolysis. The size and sequence of the duplex are such that HIV-1 RT-RNase H cuts the RNA strand close to the 3' end. The fluorescein-labeled ribonucleotide fragment readily dissociates from the complementary DNA at room temperature with immediate generation of a fluorescent signal. This assay is rapid, inexpensive, and robust, providing Z' factors of 0.8 and coefficients of variation of about 5%. The assay can be carried out both in real-time (continuous) and in "quench" modes; the latter requires only two addition steps with no washing and is thus suitable for robotic operation. Several chemical libraries totaling more than 106,000 compounds were screened with this assay in approximately 1 month.

Base Sequence↗

PubMed1996

Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes.

To improve the previous DNA/DNA nearest-neighbor parameters, thermodynamic parameters (deltaH degrees, deltaS degrees and deltaG degrees) of 50 DNA/DNA duplexes were measured. Enthalpy change of a helix initiation factor is also considered though the parameters reported recently did not contain the factor. A helix initiation factor for DNA/DNA duplex determined here was the same as that of RNA/RNA duplex (deltaG degrees(37) = 3.4 kcal/mol). The improved nearest-neighbor parameters reproduced not only these 50 experimental values used here but also 15 other experimental values obtained in different studies. Comparing deltaG degrees(37) values of DNA/DNA nearest-neighbor parameters obtained here with those of RNA/RNA and RNA/DNA, RNA/RNA duplex was generally the most stable of the three kinds of duplexes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA duplexes is sequence dependent.

Chromatography, High Pressure Liquid↗

PubMed1986

Improved free-energy parameters for predictions of RNA duplex stability.

Thermodynamic parameters for prediction of RNA duplex stability are reported. One parameter for duplex initiation and 10 parameters for helix propagation are derived from enthalpy and free-energy changes for helix formation by 45 RNA oligonucleotide duplexes. The oligomer sequences were chosen to maximize reliability of secondary structure predictions. Each of the 10 nearest-neighbor sequences is well-represented among the 45 oligonucleotides, and the sequences were chosen to minimize experimental errors in delta GO at 37 degrees C. These parameters predict melting temperatures of most oligonucleotide duplexes within 5 degrees C. This is about as good as can be expected from the nearest-neighbor model. Free-energy changes for helix propagation at dangling ends, terminal mismatches, and internal G X U mismatches, and free-energy changes for helix initiation at hairpin loops, internal loops, or internal bulges are also tabulated.

Hydrogen Bonding↗

PubMed1994

Sequence-dependent conformational heterogeneity of a hybrid DNA.RNA dodecamer duplex.

Two- and three-dimensional homonuclear NMR studies of a hybrid duplex RI, formed by annealing r(GCGCAAAACGCG) and d(CGCGTTTTGCGC) strands are described. NMR parameters, such as intra- and interresidue proton-proton NOEs and sugar proton coupling constants were analyzed with reference to those of the corresponding DNA.DNA duplex. Furthermore, spectral analyses were conducted on the basis of model structures of nucleic acid duplexes. Distinctive spectral patterns of the hybrid duplex reveal unique heterogeneous conformations which co-exist throughout the sequence and are significantly different from those of model structures of either canonical A- or B-forms. Features of an intermediate conformation were observed in the DNA and RNA strands in duplex RI, the former being more B-like and the latter more A-like. Three-dimensional NOESY-NOESY spectra were analyzed and their use was demonstrated for resolving superimposed resonances and cross peaks, especially those originating from the RNA strand. The application of a useful strategy that combines the use of 2D NMR data and the known structural information for efficient 3D spectral analyses is demonstrated.

Base Sequence↗

PubMed1999

Crystal structure of a 14 bp RNA duplex with non-symmetrical tandem GxU wobble base pairs.

Adjacent GxU wobble base pairs are frequently found in rRNA. Atomic structures of small RNA motifs help to provide a better understanding of the effects of various tandem mismatches on duplex structure and stability, thereby providing better rules for RNA structure prediction and validation. The crystal structure of an RNA duplex containing the sequence r(GGUAUUGC-GGUACC)2 has been solved at 2.1 A resolution using experimental phases. Novel refinement strategies were needed for building the correct solvent model. At present, this is the only short RNA duplex structure containing 5'-U-U-3'/3'-G-G-5' non-symmetric tandem GxU wobble base pairs. In the 14mer duplex, the six central base pairs are all displaced away from the helix axis, yielding significant changes in local backbone conformation, helix parameters and charge distribution that may provide specific recognition sites for biologically relevant ligand binding. The greatest deviations from A-form helix occur where the guanine of a wobble base pair stacks over a purine from the opposite strand. In this vicinity, the intra-strand phosphate distances increase significantly, and the major groove width increases up to 3 A. Structural comparisons with other short duplexes containing symmetrical tandem GxU or GxT wobble base pairs show that nearest-neighbor sequence dependencies govern helical twist and the occurrence of cross-strand purine stacks.

Base Pairing↗

PubMed2005

POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates.

Defects in human RecQ helicases WRN and BLM are responsible for the cancer-prone disorders Werner syndrome and Bloom syndrome. Cellular phenotypes of Werner syndrome and Bloom syndrome, including genomic instability and premature senescence, are consistent with telomere dysfunction. RecQ helicases are proposed to function in dissociating alternative DNA structures during recombination and/or replication at telomeric ends. Here we report that the telomeric single-strand DNA-binding protein, POT1, strongly stimulates WRN and BLM to unwind long telomeric forked duplexes and D-loop structures that are otherwise poor substrates for these helicases. This stimulation is dependent on the presence of telomeric sequence in the duplex regions of the substrates. In contrast, POT1 failed to stimulate a bacterial 3'-5'-helicase. We find that purified POT1 binds to WRN and BLM in vitro and that full-length POT1 (splice variant 1) precipitates a higher amount of endogenous WRN protein, compared with BLM, from the HeLa nuclear extract. We propose roles for the cooperation of POT1 with RecQ helicases WRN and BLM in resolving DNA structures at telomeric ends, in a manner that protects the telomeric 3' tail as it is exposed during unwinding.

Adenosine Triphosphatases↗

PubMed1995

Solution structure of two mismatches A.A and T.T in the K-ras gene context by nuclear magnetic resonance and molecular dynamics.

Two mismatches, one homopurine (A.A) and the other homopyrimidine (T.T), have been incorporated at the central position N of: 5'd(GCCACNAGCTC).d(GAGCTNGTGGC) in order to study nuclear magnetic resonance spectra and molecular dynamics. These duplexes constitute the sequence 29-39 of the K-ras gene coding for Gly12, a hot spot for mutation. The NMR spectra show that the duplexes are not greatly distorted by the introduction of the mismatches and their global conformation is that of a canonical B-form double helix. For both systems, no structural change is observed in the pH range 4.7-9. For the duplex containing the homopurine A.A mismatch, we propose a type of pairing involving one hydrogen bond between the amino group of one central adenine and the nitrogen N1 of the opposite adenine. For the duplex containing the mispaired T.T bases, NMR spectra recorded in H2O at 282 K indicate that these central bases are engaged in wobble pairing, involving two imino-carbonyl hydrogen bonds. For both systems two conformations with the same donor and acceptor pattern can coexist, one being obtained from the other by a 180 degrees rotation about the pseudodyadic axis. Exchange between the two forms is observed by NMR at low temperature for the T.T mispair and also inferred from NMR measurements on the A.A system. The presence of this exchange and its pathway has been investigated by molecular dynamics calculations on both systems. Distance restrained and unrestrained molecular dynamics are in very good agreement with the NMR data. The average structure for either mispair shows only small conformational change from normal B DNA. For each, a systematic pathway is observed for exchange between the two conformations.

Base Sequence↗

PubMed1995

Relative thermodynamic stability of DNA, RNA, and DNA:RNA hybrid duplexes: relationship with base composition and structure.

Fourteen oligonucleotides 8-21 nucleotides in length and their complements were synthesized as DNA and RNA. For each sequence, four kinds of duplexes, DNA:DNA, RNA:RNA, DNA:RNA, and RNA:DNA, were prepared. Twelve sequences had A.T/U content varying from 25 to 80% and dPy content in the DNA strands varying from 0 to 100%. Thermodynamic stabilities of four duplexes for each sequence were determined in solution containing 100 mM Na+, 10 mM phosphate, and 0.1 mM EDTA, pH 7.1. CD spectra and electrophoretic mobility on native polyacrylamide gel were measured for most duplexes. Quantitative correlations of hybrid stability both with deoxypyrimidine content and, at fixed dPy content, with the fraction of A.T/U in duplexes were found. We also demonstrated that hybrids with 70-80% deoxypyrimidine DNA strand and a high or moderate A.T/U fraction displayed the highest relative stability compared to their RNA counterparts. Relationships of relative intensities of CD bands at 210 nm and relative electrophoretic mobilities of hybrids with relative hybrid stability suggested that hybrid conformation varies continuously between A- and B-form and is the decisive factor in relative hybrid stability.

Base Sequence↗

PubMed2004

Sequence-dependent nucleotide dynamics revealed by intercalated ring rotation in DNA-bisnaphthalimide complexes.

Bisnaphthalimide intercalators are anti-tumour agents composed of two planar rings linked by a flexible diazanonylene chain. The intercalated rings of three bisnaphthalimide analogues complexed to DNA are found here to undergo 180 degrees rotating motions that do not affect the diazanonylene linker atoms bound to the major groove. These ring rotations are detected by NMR spectroscopy in a broad range of sequence contexts and duplex lengths. A comparative analysis of the frequency and activation energies of such excited states in different complexes and conditions indicates that these motions (i) are unrelated to drug dissociation; (ii) are a consequence of concerted, sequence-dependent nucleotide movements taking place on the millisecond time scale; and (iii) may occur inside the DNA duplexes. The rotation frequencies range from 2 to 25 s(-1) at 25 degrees C, depending on DNA composition and the size of the rotating rings. The detected nucleotide dynamics are likely to play an important role in the binding kinetics of the numerous proteins and drugs that require base unstacking when interacting with DNA.

Amides↗

PubMed2005

Sequence dependence of charge transport through DNA domains.

Here we examine the photooxidation of two kinetically fast electron hole traps, N4-cyclopropylcytosine (CPC) and N2-cyclopropylamine-guanosine (CPG), incorporated in DNA duplexes of various sequence using different photooxidants. DNA oxidation studies are carried out either with noncovalently bound [Ru(phen)(dppz)(bpy')]3+ (dppz = dipyridophenazine) and [Rh(phi)2(bpy)]3+ (phi = phenanthrenequinone diimine) or with anthraquinone tethered to DNA. Because the cyclopropylamine-substituted bases decompose rapidly upon oxidation, their efficiency of decomposition provides a measure of relative hole localization. Consistent with a higher oxidation potential for CPC versus CPG in DNA, CPC decomposes with photooxidation by [Rh(phi)2(bpy)]3+, while CPG undergoes ring-opening both with photoexcited [Rh(phi)2(bpy)]3+ and with [Ru(phen)(dppz)(bpy')]3+. Anthraquinone-modified DNA assemblies of identical base composition but different base sequence are also probed. Single and double base substitutions within adenine tracts modulate CPC decomposition. In fact, the entire sequence within the DNA assembly is seen to govern CPC oxidation, not simply the bases intervening between CPC and the tethered photooxidant. These data are reconciled in the context of a mechanistic model of conformationally gated charge transport through delocalized DNA domains. Photooxidations of anthraquinone-modified DNA assemblies containing both CPC and CPG, but with varied distances separating the modified bases, point to a domain size of at least three bases. Our model for DNA charge transport is distinguished from polaron models. In our model, delocalized domains within the base pair stack form transiently based upon sequence-dependent DNA structure and dynamics. Given these results, DNA charge transport is indeed remarkably sensitive to DNA sequence and structure.

Base Sequence↗

PubMed2003

Kissing complex-mediated dimerisation of HIV-1 RNA: coupling extended duplex formation to ribozyme cleavage.

Initiation of retroviral genomic RNA dimerisation is mediated by the mutual interaction of the dimerisation initiation site (DIS) stem-loops near to the 5' end of the RNA. This process is thought to involve formation of a transient 'kissing' complex over the self-complementary loop bases, which then refolds into a more stable extended interaction. We have developed a novel experimental system that allows us to clearly detect the extended duplex in vitro. Ribozyme sequences were incorporated into or adjacent to the type 1 human immunodeficiency virus DIS stem, leading to the formation of a functional ribozyme only in the extended duplex conformer. Here we show that extended duplex formation results in ribozyme cleavage, thus demonstrating the double-stranded nature of the extended complex and confirming that refolding occurs via melting of the DIS stems. Loop complementarity is essential for extended duplex formation but no sequence requirements for the loops were observed. Efficiency of extended duplex formation is dependent on the strength of the loop-loop interaction, temperature, the magnesium concentration and is strongly accelerated by the viral nucleocapsid protein NCp7. Our ribozyme-coupled approach should be applicable to the analyses of other refolding processes involving RNA loop-loop interactions.

Base Sequence↗

PubMed1994

The reactivity of 4,5',8-trimethylpsoralen with oligonucleotides containing AT sites.

Pyrimidine bases of duplex DNA, of appropriate sequence context, are photoreactive toward 4,5',8-trimethylpsoralen in the presence of long-wavelength UV light. It is generally believed that a 5'-AT site is less photoreactive with psoralen than a 5'-TA site. We have compared the reactivities of these two sites using oligonucleotide duplexes of different sequence context and found that 5'-TA and 5'-AT sites are equally reactive in certain sequences. The presence of alternating pyrimidine and purine (5'-PyATPu-3') bases in oligonucleotide duplexes optimizes the reactivity of 4,5',8-trimethylpsoralen in the 5'-AT sites.

Adenine↗