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PubMed1999

Stability of G,A triple helices.

In this work we selected double-stranded DNA sequences capable of forming stable triplexes at 20 or 50 degrees C with corresponding 13mer purine oligonucleotides. This selection was obtained by a double aptamer approach where both the starting sequences of the oligonucleotides and the target DNA duplex were random. The results of selection were confirmed by a cold exchange method and the influence of the position of a 'mismatch' on the stability of the triplex was documented in several cases. The selected sequences obey two rules: (i) they have a high G content; (ii) for a given G content the stability of the resulting triplex is higher if the G residues lie in stretches. The computer simulation of the Mg2+, Na+and Cl-environment around three triplexes by a density scaled Monte Carlo method provides an interpretation of the experimental observations. The Mg2+cations are statistically close to the G N7 and relatively far from the A N7. The presence of an A repels the Mg2+from adjacent G residues. Therefore, the triplexes are stabilized when the Mg2+can form a continuous spine on G N7.

Base Sequence↗

PubMed1996

An antisense/target RNA duplex or a strong intramolecular RNA structure 5' of a translation initiation signal blocks ribosome binding: the case of plasmid R1.

Antisense RNAs in prokaryotic systems often inhibit translation of mRNAs. In some cases, this involves sequestration of Shine-Dalgarno (SD) sequences and start codons. In other cases, antisense/target RNA duplexes do not overlap these signals, but form upstream. We have performed toeprinting analyses on repA mRNA of plasmid R1, both free and in duplex with the antisense RNA, CopA. An intermolecular RNA duplex 2 nt upstream of the tap SD prevents ribosome binding. An intrastrand stem-loop at this location yields the same inhibition. Thus, stable secondary structures immediately upstream of the tap SD sequence inhibit translation, as shown by toeprinting in vitro and repA-lacZ expression in vivo. Previous work showed that repA (initiator protein) expression requires tap (leader peptide) translation. Toeprinting data confirm that the tap ribosome binding site (RBS) is accessible, whereas the repA RBS, which is sequestered by a stable stem-loop, is weakly recognized by the ribosome. Truncated CopA RNA (CopI) is unable to pair completely with target RNA, but proceeds normally to a kissing intermediate. This mutant RNA species inhibits repA expression in vivo. By a kinetic toeprint inhibition protocol, we have shown that the structure of the kissing complex is sufficient to sterically prevent ribosome binding. These results are discussed in comparison with the effect of RNA structures elsewhere in the ribosome-binding region of an mRNA.

Bacterial Proteins↗

PubMed1992

Highly conserved repetitive DNA sequences are present at human centromeres.

Highly conserved repetitive DNA sequence clones, largely consisting of (GGAAT)n repeats, have been isolated from a human recombinant repetitive DNA library by high-stringency hybridization with rodent repetitive DNA. This sequence, the predominant repetitive sequence in human satellites II and III, is similar to the essential core DNA of the Saccharomyces cerevisiae centromere, centromere DNA element (CDE) III. In situ hybridization to human telophase and Drosophila polytene chromosomes shows localization of the (GGAAT)n sequence to centromeric regions. Hyperchromicity studies indicate that the (GGAAT)n sequence exhibits unusual hydrogen bonding properties. The purine-rich strand alone has the same thermal stability as the duplex. Hyperchromicity studies of synthetic DNA variants indicate that all sequences with the composition (AATGN)n exhibit this unusual thermal stability. DNA-mobility-shift assays indicate that specific HeLa-cell nuclear proteins recognize this sequence with a relative affinity greater than 10(5). The extreme evolutionary conservation of this DNA sequence, its centromeric location, its unusual hydrogen bonding properties, its high affinity for specific nuclear proteins, and its similarity to functional centromeres isolated from yeast suggest that this sequence may be a component of the functional human centromere.

Animals↗

PubMed2001

The crystal structures of psoralen cross-linked DNAs: drug-dependent formation of Holliday junctions.

The single-crystal structures are presented for two DNA sequences with the thymine bases covalently cross-linked across the complementary strands by 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT). The HMT-adduct of d(CCGCTAGCGG) forms a psoralen-induced Holliday junction, showing for the first time the effect of this important class of chemotheraputics on the structure of the recombination intermediate. In contrast, HMT-d(CCGGTACCGG) forms a sequence-dependent junction. In both structures, the DNA duplex is highly distorted at the thymine base linked to the six-member pyrone ring of the drug. The psoralen cross-link defines the intramolecular interactions of the drug-induced junction, while the sequence-dependent structure is nearly identical to the native Holliday junction of d(CCGGTACCGG) alone. The two structures contrast the effects of drug- and sequence-dependent interactions on the structure of a Holliday junction, suggesting a role for psoralen in the mechanism to initiate repair of psoralen-lesions in mammalian DNA.

Animals↗

PubMed2001

Base sequence effects in bending induced by bulky carcinogen-DNA adducts: experimental and computational analysis.

The covalent binding of bulky mutagenic or carcinogenic compounds to DNA can lead to bending, which could significantly alter the interactions of DNA with critical replication and transcription proteins. The impact of adducts derived from the highly reactive bay region enantiomeric (+)- and (-)-anti-7,8-diol-9,10-epoxide derivatives of benzo[a]pyrene (BPDE) are of interest because the (+)-7R,8S,9S,10R-anti-BPDE enantiomer is highly tumorigenic in rodents, while the (-)-7S,8R,9R,10S-anti-BPDE enantiomer is not. Both (+)- and (-)-anti-BPDE bind covalently with DNA predominantly by trans addition at the exocyclic amino group of guanine to yield 10S (+)- and 10R (-)-trans-anti-[BP]-N(2)-dG adducts. We have synthesized a number of different oligonucleotides with single (+)- and (-)-trans-anti-[BP]-N(2)-dG adducts (G) in the base sequence context XG*Y, where X and Y are different DNA bases. The G* residues were positioned at or close to the center of 11 base pair ( approximately 1 helical turn) or 16 base pair ( approximately 1.5 turns) duplexes. All bases, except for X and Y and their partners, were identical. These sequences were self-ligated with T4 ligase to form multimers that yield a ladder of bands upon electrophoresis in native polyacrylamide gels. The extent of bending in each oligonucleotide was assessed by monitoring the decrease in gel mobilities of these linear, self-ligated oligomers, relative to unmodified oligonucleotides of the same base sequence. The extent of global bending was then estimated using a sequence-specific three-dimensional model from which the values of the base-pair step parameter roll adjacent to the lesion site could be extracted. We find that (+)-trans-anti-[BP]-N(2)-dG adducts are considerably more bent than the (-) isomers regardless of sequence and that A-T base pairs flanking the [BP]-N(2)-dG lesion site allow for local flexibility consistent with adduct conformational heterogeneity. Interestingly, the fit of computed versus observed gel mobilities using classical reptation treatments requires enhancement of unmodified DNA flexibility in gels, compared to aqueous salt solution. The differences in bending between the two stereoisomeric adduct duplexes and the observed base sequence context effects may play a significant role in the differential processing of these lesions by cellular replication, transcription, and repair enzymes.

Algorithms↗

PubMed1987

DNA polymorphism of MHC III genes in inbred and wild mouse strains.

Genes encoding the second component (C2), factor B, and complement protein C4 and Slp (sex-limited protein) are members of the major histocompatibility complex class III gene cluster. In this report we describe isolation of a mouse C2 cDNA clone and its use together with factor B and C4 cDNA clones to examine the S region in a panel of 42 haplotypes in laboratory and wild mice representing 5 species and subspecies of Mus. Conservation of the C2 factor B gene duplex was evidenced by relatively limited polymorphism associated with speciation and nucleotide sequence homology between mouse and human C2 and factor B. The C4-Slp gene duplex, on the other hand, showed extensive polymorphism by DNA blot analysis. This polymorphism correlated poorly with the C2/factor B restriction fragment length polymorphism, suggesting independent evolution of these two segments of the S region. Taken together, these data will be of particular importance in studies of mouse strains with abnormal regulation of immune effector systems since the class III gene products are essential for activation of the complement cascade.

Amino Acid Sequence↗

PubMed1989

A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA.

A method is presented for choosing optimal oligodeoxyribonucleotides as probes for filter hybridization, primers for sequencing, or primers for DNA amplification. Three main factors that determine the quality of a probe are considered: stability of the duplex formed between the probe and target nucleic acid, specificity of the probe for the intended target sequence, and self-complementarity. DNA duplex stability calculations are based on the nearest-neighbor thermodynamic values determined by Breslauer et al. [Proc. Natl. Acad. Sci. U.S.A. (1986), 83: 3746]. Temperatures of duplex dissociation predicted by the method described here were within 0.4 degrees C of the values obtained experimentally for ten oligonucleotides. Calculations for specificity of the probe and its self-complementarity are based on a simple dynamic algorithm.

Base Sequence↗

PubMed2002

Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets.

Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.

Base Pair Mismatch↗

PubMed2005

Site-selective RNA cleavage by DNA bearing a base pair-mimic nucleoside.

We have synthesized the deoxyadenosine derivative tethering a phenyl group (X), which mimics the Watson-Crick A/T base pair. The RNA/DNA hybrid duplexes containing X in the middle of the DNA sequence showed a similar thermal stability regardless of the ribonucleotide species (A, G, C, or U) opposite to X, probably because of the phenyl group stacking inside of the duplex accompanied by the opposite ribonucleotide base flipped in an extrahelical position. The RNA strand hybridized with the DNA strand bearing X was cleaved on the 3'-side of the ribonucleotide opposite to X in the presence of MgCl2, and the RNA sequence to be cleaved was not restricted. The site-specific RNA hydrolysis suggests that the DNA strand bearing X has the advantage of the site-selective base flipping in the target sequence and the development of a "universal deoxyribozyme" to exclusively cleave a target RNA sequence.

Adenosine↗

PubMed1996

RNA editing.

RNA editing is a term describing a variety of novel mechanisms for the modification of nucleotide sequences of RNA transcripts in different organisms. These editing events include (a) the U-insertion and -deletion type of editing found in the mitochondrion of kinetoplastid protozoa, (b) the C-insertion editing found in the mitochondrion of Physarum, (c) the C-to-U substitution editing of the mammalian apoB mRNA, (d) a similar C-to-U substitution editing of mRNAs in higher plant mitochondria and chloroplasts and in tRNAs of marsupials and rats, (e) a diverse nucleotide substitution editing of tRNAs in Acanthomoeba mitochondria, and (f) the A-to-I type of editing found in the mammalian glutamate receptor subunits. These diverse phenomena involve several different enzymatic mechanisms. In several cases, duplex RNAs with internal or external guide sequences help determine the site specificity of editing. The A-to-I editing observed in RNAs encoding non-NMDA glutamate receptor subunits may be due to the actions of a double-stranded RNA-specific adenosine deaminase that is widespread in higher organisms. Although the function of many RNA editing events is unclear, the biological importance of RNA editing in other systems may prove as significant as the nucleotide modifications regulating the cation selectivity and electrophysiological profiles elaborated by non-NMDA glutamate receptors in the mammalian brain.

Animals↗

PubMed2002

Characterization of RNA determinants recognized by the arginine- and proline-rich region of Us11, a herpes simplex virus type 1-encoded double-stranded RNA binding protein that prevents PKR activation.

The herpes simplex virus Us11 gene product inhibits activation of the cellular PKR kinase and associates with a limited number of unrelated viral and cellular RNA molecules via a carboxyl-terminal 68-amino-acid segment rich in arginine and proline. To characterize the determinants underlying the recognition of an RNA target by Us11, we employed an in vitro selection technique to isolate RNA ligands that bind Us11 with high affinity from a population of molecules containing an internal randomized segment. Binding of Us11 to these RNA ligands is specific and appears to occur preferentially on conformational isoforms that possess a higher-order structure. While the addition of unlabeled poly(I. C) reduced binding of Us11 to a selected radiolabeled RNA, single-stranded homopolymers were not effective competitors. Us11 directly associates with poly(I. C), and inclusion of an unlabeled selected RNA in the reaction reduces poly(I. C) binding, while single-stranded RNA homopolymers have no effect. Finally, Us11 binds to defined, double-stranded RNA (dsRNA) molecules that exhibit greater sequence complexity. Binding to these dsRNA perfect duplexes displays a striking dependence on length, as 39-bp or shorter duplexes do not bind efficiently. Furthermore, this interaction is specific for dsRNA as opposed to dsDNA, implying that the Us11 RNA binding domain can distinguish nucleic acid duplexes containing 2' hydroxyl groups from those that do not. These results establish that Us11 is a dsRNA binding protein. The arginine- and proline-rich Us11 RNA binding domain is unrelated to known dsRNA binding elements and thus constitutes a unique recognition motif that interacts with dsRNA. The ability of Us11 to bind dsRNA may be important for inhibiting activation of the cellular PKR kinase in response to dsRNA.

Arginine↗

PubMed2005

Conformational determinants of tandem GU mismatches in RNA: insights from molecular dynamics simulations and quantum mechanical calculations.

Structure and energetic properties of base pair mismatches in duplex RNA have been the focus of numerous investigations due to their role in many important biological functions. Such efforts have contributed to the development of models for secondary structure prediction of RNA, including the nearest-neighbor model. In RNA duplexes containing GU mismatches, 5'-GU-3' tandem mismatches have a different thermodynamic stability than 5'-UG-3' mismatches. In addition, 5'-GU-3' mismatches in some sequence contexts do not follow the nearest-neighbor model for stability. To characterize the underlying atomic forces that determine the structural and thermodynamic properties of GU tandem mismatches, molecular dynamics (MD) simulations were performed on a series of 5'-GU-3' and 5'-UG-3' duplexes in different sequence contexts. Overall, the MD-derived structural models agree well with experimental data, including local deviations in base step helicoidal parameters in the region of the GU mismatches and the model where duplex stability is associated with the pattern of GU hydrogen bonding. Further analysis of the simulations, validated by data from quantum mechanical calculations, suggests that the experimentally observed differences in thermodynamic stability are dominated by GG interstrand followed by GU intrastrand base stacking interactions that dictate the one versus two hydrogen bonding scenarios for the GU pairs. In addition, the inability of 5'-GU-3' mismatches in different sequence contexts to all fit into the nearest-neighbor model is indicated to be associated with interactions of the central four base pairs with the surrounding base pairs. The results emphasize the role of GG and GU stacking interactions on the structure and thermodynamics of GU mismatches in RNA.

Base Pair Mismatch↗

PubMed1995

A DNA sequencing strategy that requires only five bases of known terminal sequence for priming.

We have previously reported an enhanced version of sequencing by hybridization (SBH), termed positional SBH (PSBH). PSBH uses partially duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target allow us to reduce the probe sizes required to 5-base single-stranded overhangs. Here we demonstrate the use of PSBH to capture relatively long single-stranded DNA targets and perform standard solid-state Sanger sequencing on these primer-template complexes without ligation. Our results indicate that only 5 bases of known terminal sequence are required for priming. In addition, the partially duplex probes have the ability to capture their specific target from a mixture of five single-stranded targets with different 3'-terminal sequences. This indicates the potential utility of the PSBH approach to sequence mixtures of DNA targets without prior purification.

Base Sequence↗

PubMed1998

High resolution HLA class I typing by reference strand mediated conformation analysis (RSCA).

We describe the application of RSCA for the high resolution typing of alleles encoded within the highly polymorphic HLA class I system. RSCA differs from other sequence based typing methodologies in that the HLA type is assigned on the basis of differences in DNA conformation between different HLA alleles. HLA class I locus specific PCR was performed on the sample to be HLA typed and the PCR product mixed with a fluorescent labelled locus specific reference strand. DNA molecules were allowed to reanneal and the duplexes formed were separated by non-denaturing PAGE. Only duplexes which possess the fluorescent labelled reference strand were detected using an automated DNA sequencer. The mobilities of duplexes formed for 131 HLA class I alleles have been calculated and HLA type assigned. The use of a laser scanning system for accurate detection of migrating DNA, in combination with specific software for data analysis and correction of gel variablility, make RSCA sufficiently robust to detect alleles differing by as little as one nucleotide, resulting in a simple and automated technique for high resolution HLA typing.

Alleles↗

PubMed1995

An intramolecular triplex in the human gamma-globin 5'-flanking region is altered by point mutations associated with hereditary persistence of fetal hemoglobin.

The properties of an intramolecular triplex formed in vitro at the 5'-flanking region of the human gamma-globin genes were studied by chemical and physical probes. Chemical modifications performed with osmium tetroxide, chloroacetaldehyde, and diethyl pyrocarbonate revealed the presence of non-paired nucleotides on the "coding strand" at positions -209 through -217. These reactivities were induced by negative supercoiling, low pH, and magnesium ions. Downstream point mutations associated with hereditary persistence of fetal hemoglobin (HPFH) altered the extent of the modifications and some of the patterns. Specifically, C-202-->G and C-202-->T significantly decreased the reactivities, whereas the patterns were increased and altered in the T-198-->C. C-196-->T and C-195-->G caused local decreases in reactivity. Modifications at the upstream flanking duplex were modulated by the composition of the vector sequence. In summary, our data indicates the formation of an intramolecular triplex between nucleotides -209 to -217 of the "non-coding strand" and the downstream sequence containing the HPFH mutations. All of the HPFH point mutations altered the structure. More than one sequence alignment is possible for each of the triplexes. In addition, a consequence of some of the point mutations may be to facilitate slippage of the third strand relative to the Watson-Crick duplex.

Acetaldehyde↗

PubMed2002

Microchip bioprocessor for integrated nanovolume sample purification and DNA sequencing.

A microfabricated electrophoretic bioprocessor for integrated DNA sequencing sample desalting, template removal, preconcentration, and CE analysis is presented. A low-viscosity gel capture matrix, containing an acrylamide-copolymerized oligonucleotide complementary to the 20-base sequence directly 3' of the M13-40 universal forward priming site, is introduced into the 60-nL capture chamber. Unpurified DNA sequencing reaction products are electrophoretically driven through the chamber; extension products hybridize to the matrix, while contaminating buffering ions, Cl-, excess primer, and template DNA are unretained. Purification under optimized conditions is complete in only 120 s (binding temperature 50 degrees C, driving voltage 250 V). High-speed, integrated sequencing analysis is accomplished by releasing the gel-purified duplex at 67 degrees C and directly injecting onto a 15.9-cm effective length CE microchannel. Electrophoretic resolution of the sequencing products is complete in 32 min, producing a total of 560 bp with phred quality q > or = 20 (accuracy > or = 99%). This fully integrated nanoliter process decreases the purification time approximately 10-fold and the process volume approximately 100-fold while providing state-of-the-art sequencing results.

Algorithms↗

PubMed1994

Model for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix.

In large structured RNAs, RNA hairpins in which the strands of the duplex stem are connected by a tetraloop of the consensus sequence 5'-GNRA (where N is any nucleotide, and R is either G or A) are unusually frequent. In group I introns there is a covariation in sequence between nucleotides in the third and fourth positions of the loop with specific distant base pairs in putative RNA duplex stems: GNAA loops correlate with successive 5'-C-C.G-C base pairs in stems, whereas GNGA loops correlate with 5'-C-U.G-A. This has led to the suggestion that GNRA tetraloops may be involved in specific long-range tertiary interactions, with each A in position 3 or 4 of the loop interacting with a C-G base pair in the duplex, and G in position 3 interacting with a U-A base pair. This idea is supported experimentally for the GAAA loop of the P5b extension of the group I intron of Tetrahymena thermophila and the L9 GUGA terminal loop of the td intron of bacteriophage T4 (ref. 4). NMR has revealed the overall structure of the tetraloop for 12-nucleotide hairpins with GCAA and GAAA loops and models have been proposed for the interaction of GNRA tetraloops with base pairs in the minor groove of A-form RNA. Here we describe the crystal structure of an intermolecular complex between a GAAA tetraloop and an RNA helix. The interactions we observe correlate with the specificity of GNRA tetraloops inferred from phylogenetic studies, suggesting that this complex is a legitimate model for intramolecular tertiary interactions mediated by GNRA tetraloops in large structured RNAs.

Crystallography↗

PubMed1981

All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus.

In hypotrichous ciliates, all of the macronuclear DNA is in the form of low molecular weight molecules with an average size of approximately 2200 base pairs. Total macronuclear DNA from four hypotrichs has been shown to have inverted terminal repeats by direct sequence analysis. In Oxytricha nova, Oxytricha sp., and Stylonychia pustulata, this terminal sequence may be written as 5'-C4A4C4A4C4 ... 3'-G4T4G4T4G4T4G4T4G4 ... In Euplotes aediculatus, the sequences is similar but differs in the lengths of the duplex region (28 base pairs) and of the putative 3' extension (14 base pairs). Also in Euplotes, a second common sequence of 5 base pairs (A-A-C-T-T-T-T-G-A-A) occurs internal to the terminal repeat and a 17-base-pair heterogeneous region: 5'-C4A4C4A4C4A4C4(X)17T-T-G-A-A ... 3'-G2T4G4T4G4T4G4T4G4T4G4(X)17A-A-C-T-T ... The length of the terminal repeat sequence for O. nova was confirmed in cloned macronuclear DNA molecules.

Animals↗