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PubMed2001

Stabilization factors affecting duplex formation of peptide nucleic acid with DNA.

Peptide nucleic acid (PNA) is an oligonucleotide analogue in which the sugar-phosphate backbone is replaced by an N-(2-aminoethyl)glycine unit to which the nucleobases are attached. We investigated the thermodynamic behavior of PNA/DNA hybrid duplexes with identical nearest neighbors but with different sequences and chain lengths (5, 6, 7, 8, 10, 12, and 16 mers) to reveal whether the nearest-neighbor model is valid for the PNA/DNA duplex stability. CD spectra of 6, 7, and 8 mer PNA/DNA duplexes showed similar signal, while 10, 12, and 16 mer duplexes did not. The average difference in Delta G degrees (37) for short PNA/DNA duplexes with identical nearest-neighbor pairs was only 3.5%, whereas that of longer duplexes (10, 12, and 16 mers) was 16.4%. Therefore, the nearest-neighbor model seems to be useful at least for the short PNA/DNA duplexes. Thermodynamics of PNA/DNA duplexes containing 1--3 bulge residues were also studied. While the stability of the 12 mer DNA/DNA duplex decreased as the number of bulge bases increases, the number of bulge bases in PNA/DNA unchanged the duplex stability. Thus, the influence of bulge insertion in the PNA/DNA duplexes is different from that of a DNA/DNA duplex. This might be due to the different base geometry in a helix which may potentially make hydrogen bonds in a base pair and stacking interaction unfavorable compared with DNA/DNA duplexes.

Base Composition↗

PubMed2004

Identification of regions in multiple sequence alignments thermodynamically suitable for targeting by consensus oligonucleotides: application to HIV genome.

BACKGROUND: Computer programs for the generation of multiple sequence alignments such as "Clustal W" allow detection of regions that are most conserved among many sequence variants. However, even for regions that are equally conserved, their potential utility as hybridization targets varies. Mismatches in sequence variants are more disruptive in some duplexes than in others. Additionally, the propensity for self-interactions amongst oligonucleotides targeting conserved regions differs and the structure of target regions themselves can also influence hybridization efficiency. There is a need to develop software that will employ thermodynamic selection criteria for finding optimal hybridization targets in related sequences. RESULTS: A new scheme and new software for optimal detection of oligonucleotide hybridization targets common to families of aligned sequences is suggested and applied to aligned sequence variants of the complete HIV-1 genome. The scheme employs sequential filtering procedures with experimentally determined thermodynamic cut off points: 1) creation of a consensus sequence of RNA or DNA from aligned sequence variants with specification of the lengths of fragments to be used as oligonucleotide targets in the analyses; 2) selection of DNA oligonucleotides that have pairing potential, greater than a defined threshold, with all variants of aligned RNA sequences; 3) elimination of DNA oligonucleotides that have self-pairing potentials for intra- and inter-molecular interactions greater than defined thresholds. This scheme has been applied to the HIV-1 genome with experimentally determined thermodynamic cut off points. Theoretically optimal RNA target regions for consensus oligonucleotides were found. They can be further used for improvement of oligo-probe based HIV detection techniques. CONCLUSIONS: A selection scheme with thermodynamic thresholds and software is presented in this study. The package can be used for any purpose where there is a need to design optimal consensus oligonucleotides capable of interacting efficiently with hybridization targets common to families of aligned RNA or DNA sequences. Our thermodynamic approach can be helpful in designing consensus oligonucleotides with consistently high affinity to target variants in evolutionary related genes or genomes.

Base Sequence↗

PubMed1994

A universal nucleoside for use at ambiguous sites in DNA primers.

A non-discriminatory base analogue, or universal base, would be an invaluable component of oligonucleotide probes and primers for solving the design problems that arise as a result of the degeneracy of the genetic code, or when only fragmentary peptide sequence data are available. We have designed an alternative to previous universal nucleoside candidates, a new analogue, 1-(2'-deoxy-beta-D-ribofuranosyl)-3-nitropyrrole (designated M; Fig. 1), which maximizes stacking while minimizing hydrogen-bonding interactions without sterically disrupting a DNA duplex. Oligonucleotides containing M at several sites were used as primers for sequencing and the polymerase chain reaction. The sequencing primer d(5'-CGT AAM CAM AAM ACM AT-3') is as effective as the exact match d(5'-CGT AAT CAG AAA ACA AT-3'). It is also possible to sequence using a primer containing M at several contiguous positions, for example d(5'-CGT AAT MMM MMM MMM AT-3'). Melting curves show that duplexes formed on hybridization of the sequences d(5'-CCT TTT TMT TTT TGG-3') and d(5'-CCA AAA AXA AAA AGG-3'), where X is A, C, G or T, melted at a lower temperature than the corresponding duplexes containing only d(A.T) and d(C.G) base pairs, but showing little variation among different X bases (Tm range 3 degrees C).

Animals↗

PubMed1993

The 3'-terminal end (NCCA) of tRNA determines the structure and stability of the aminoacyl acceptor stem.

We have done a systematic study on the contribution of the single-stranded NCCA end (where N is any nucleotide) to the stability of the aminoacyl stem of tRNA. A 7-bp RNA duplex with the single-strand ACCA 3' terminus derived from the aminoacyl stem of Escherichia coli tRNA(Ala) and several chemically synthesized sequence variants are characterized by proton NMR and thermodynamic parameters. The single-stranded 3' terminus noticeably stabilizes the duplex in a sequence-dependent manner. Though the largest contribution to the stability gain due to the ACCA end is provided by the first dangling 3' nucleotide, the influence of even the fourth nucleotide is measurable. The nature of the N73 discriminator base influences the stem structure and stability, which may be important for the recognition of tRNA by aminoacyl-tRNA synthetase. The stepwise attachment of the nucleotides to the 3' tail improves the stacking of the unpaired bases over the helix stem. Hence, the ACCA end appears to be structured. Replacing Mg2+ with Mn2+ causes broadening of certain imino proton peaks in the NMR spectrum, indicating a specific divalent metal ion binding site in the vicinity of the major identity element of the duplex (G3-U70) that is required for its recognition by the Ala-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗

PubMed2006

Synthesis and pairing properties of alpha-tricyclo-DNA.

We describe the synthesis of the phosphoramidite building blocks of alpha-tricyclo-DNA (alpha-tc-DNA) covering all four natural bases, starting from the already known corresponding alpha-tc-nucleosides. These building blocks were used for the preparation of three alpha-tc-oligonucleotide 10-mers representing a homopurine, a homopyrimidine, and a mixed purine/pyrimidine base sequence. The base-pairing properties with complementary parallel and antiparallel oriented DNA and RNA were studied by UV-melting analysis and CD spectroscopy. We found that alpha-tc-DNA binds preferentially to parallel nucleic acid complements through Watson-Crick duplex formation, with a preference for RNA over DNA. In comparison with natural DNA, alpha-tc-DNA shows equal to enhanced affinity to RNA and also pairs to antiparallel DNA or RNA complements, although with much lower affinity. In the mixed-base sequence these antiparallel duplexes are of the reversed Watson-Crick type, while in the homopurine/homopyrimidine sequences Hoogsteen and/or reversed Hoogsteen pairing is observed. Antiparallel duplex formation of two alpha-tc-oligonucleotides was also observed, although the thermal stability of this duplex was surprisingly low. The base-pairing properties of alpha-tc-DNA are discussed in the context of alpha-DNA, alpha-RNA, and alpha-LNA.

Base Pairing↗

PubMed1991

Sequence requirements for protein-primed DNA replication of bacteriophage PRD1.

In vitro studies have demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism. No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA. We have determined the minimal origin of replication by analyzing the template activity of various deletion derivatives. Our results showed that the terminal 20 base-pairs of ITR are required for efficient in vitro DNA replication. We have found that, within the minimal replication origin region, there are complementary sequences. A site-specific mutagenesis analysis showed that most of the point mutations in the complementary sequences markedly reduced the template activity. The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3'-terminal C residue is required for the initiation of PRD1 DNA replication in vitro.

Bacteriophages↗

PubMed2005

8-vinyl-deoxyadenosine, an alternative fluorescent nucleoside analog to 2'-deoxyribosyl-2-aminopurine with improved properties.

We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2'-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson-Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP.

Deoxyadenosines↗

PubMed1979

DNA sequence organization in the alga Euglena gracilis.

The sequence organization of nuclear DNA in the single-celled alga Euglena gracilis has been studied by a combination of techniques: (1) the comparison of the reassociation kinetics of DNA fragments 300, 2000 and 8100 nucleotides long; (2) the reassociation of 32P-labeled DNA fragments of various lengths with driver fragments 300 nucleotides long; (3) the hyperchromicity of DNA structures formed by the reassociation of repetitive sequences; (4) and the direct measurement of the size of the duplex regions of reassociated repetitive DNA resistant to S1 nuclease. The single copy DNA sequences are approximately 1500 nucleotide pairs long and are interspersed with repetitive DNA sequences. The repetitive DNA, consisting of both highly repetitive and middle repetitive sequences, consists of one fraction of nucleotide sequences (0.67) with an average size of 4900 nucleotide pairs and a second fraction (0.33) with an average size of 1000 nucleotide pairs, 34% of the DNA consists of foldback sequences which are present on 45% of the DNA 4000 nucleotides long.

Base Sequence↗

PubMed1985

Presence of retrovirus reverse transcriptase-related gene sequences in avian cells lacking endogenous avian leukosis viruses.

Using a molecularly cloned viral DNA probe representing the entire avian sarcoma virus (ASV) reverse transcriptase (pol) gene, we have detected related sequences in DNA preparations from two avain species, ev- chickens and Japanese quail, previously demonstrated to lack all endogenous avain leukosis viruses. Nucleotide sequence homology was detected only when hybridization conditions, which allowed the formation of stable duplexes with as much as 30% base mismatch, were used. No sequence homology could be detected when stringent hybridization conditions were used. Nucleotide sequence analysis of a clone representing the major pol-specific EcoRI restriction fragment from ev- chicken embryo fibroblasts revealed DNA homology as high as 72% and implied amino acid homology as high as 82% when compared to the sequence of the ASV strain Prague C pol gene. These data reveal the presence of retroviral pol gene sequences in avian cell lines that lack endogenous retrovirus sequences, suggesting that a reverse transcriptase-related gene exists in these cells as either part of a more distantly evolved retrovirus or a cellular gene.

Amino Acid Sequence↗

PubMed1988

An extended RNA/RNA duplex structure within the coding region of mRNA does not block translational elongation.

RNA/RNA duplex formation involving the 5'untranslated region of a mRNA can efficiently block translation. Here we investigated the effect on translation of an RNA/RNA duplex between part of the coding region and sequences of the 3'untranslated region of lysozyme mRNA. A cDNA was constructed which contained 2 identical sequences of 150 nucleotides, one of which was an inverted repeat of the other. Cell-free transcription of this cDNA with T7 RNA polymerase resulted in a mRNA with an extended RNA/RNA duplex within the coding region. The presence of the double stranded structure was confirmed by the accessibility of complementary oligonucleotides to this region. mRNA was cleaved by RNaseH, endogenous to the wheat germ lysate, when hybridization of a complementary oligonucleotide occurred outside but not within the predicted double stranded structure. When this mRNA was translated in a cell-free wheat germ translation system, the translation product was found to be of the size of full-length prelysozyme and not arrested. We conclude that the extend of a secondary structure within the coding region of a mRNA does not restrict the ability of the ribosome to translate this mRNA efficiently. Our data are consistent with the presence of an activity unwinding RNA/RNA duplexes, which is associated with the translating ribosome.

DNA↗

PubMed2003

Evoked potentials to auditory movement sensation in duplex perception.

OBJECTIVE: The purpose of this study was to examine the processing of auditory movement sensation accompanying duplex perception in binaural hearing. METHODS: Stimuli were formant transitions (presented to the front, left or right of the subject) and base (presented to the front), that fused to result in vowel-consonant-vowel (V-C-V) sequences /aga/ and /ada/. An illusion of auditory movement (duplex sensation) accompanied the fusion of these V-C-V sequences when the spatial locations of the formant transitions and base were different. Ten right-handed, adult, native Hebrew speakers discriminated each fused stimulus, and the brain potentials associated with performance of the task were recorded from 21 electrodes. The processing of auditory movement was studied by a factorial design (ANOVA) and statistical non-parametric mapping (SnPM) of low resolution electromagnetic tomography (LORETA) images of the net-fusion response. Brain regions implicated in auditory movement processing were expected to be associated with the lateralized formant location, which gave rise to duplex perception. In addition, the time-course of significant activation in brain areas that differentiated between fusion conditions was determined. RESULTS: The posterior parietal, anterior cingulate and premotor cortices were found to be implicated in duplex processing. Auditory cortex involvement was also evident, and together with the latter two brain regions was affected by right-ear advantage. CONCLUSIONS: Duplex perception resulting from fusion of spatially separate sounds forming an auditory object results in activation of a network of brain regions reflecting enhanced allocation of attention and the effect of language processing.

Acoustic Stimulation↗

PubMed1989

Apparent sequence preference in cleavage of linear B-DNA by the Cu(II):thiol system.

Cleavage of a linear tyr T fragment of duplex DNA by Cu(II):thiol combinations occurs with apparent sequence preference. The sequence selectivity was unaffected by the size, chirality or substitution pattern of the monothiol partner, presumably indicating that the Cu(II):RSH system is probing some local conformational/dynamic aspect of the DNA.

3-Mercaptopropionic Acid↗

PubMed1993

Identification of important residues within the putative nucleoside binding site of HSV-1 thymidine kinase by random sequence selection: analysis of selected mutants in vitro.

Random sequence mutagenesis in conjunction with genetic complementation was used to map the function of amino acid residues within the putative nucleoside binding site of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK). Six codons of the putative nucleoside binding site of the HSV-1 tk were substituted by a duplex of extended oligonucleotides containing 20% random sequences. Approximately 260 mutants were screened for the ability to genetically complement a TK-deficient Escherichia coli. Of those screened, 32% conferred TK activity. Approximately 60% of the TK positive clones contained single amino acid changes, 23% contained double changes, and 13.4% encoded the wild-type TK amino acid sequence. A small percentage of clones, 2.4% and 1.2%, contained triple or quadruple alterations, respectively. Three residues (D162, H163, and R164) appeared to be highly conserved especially with regard to the type of residues able to substitute. Secondary screening results indicated that several of the mutants had higher affinities for acyclovir and/or 3'-azido-3'-deoxythymidine than thymidine in complementation assays. In addition, a number of clones were unable to form colonies on selection medium at elevated temperatures (42 degrees C). Eight selected mutants were subcloned into an in vitro transcription vector and the derived transcripts used to program a rabbit reticulocyte lysate cell-free translation system. Biologically active translation products were then analyzed in vitro for thymidine kinase activity, for thermal stability, and for the ability to phosphorylate selected nucleoside analogues. Two of the eight mutants had an elevated thymidine kinase activity, two were significantly thermolabile, and three exhibited enhanced efficiency in phosphorylation of nucleoside analogues.

Animals↗

PubMed2000

Crystallization and preliminary X-ray diffraction analysis of MspI restriction endonuclease in complex with its cognate DNA.

The MspI restriction endonuclease is a type II restriction enzyme. Unlike all other restriction enzymes with known structures, MspI recognizes the palindromic tetranucleotide sequence 5'-C/CGG and cleaves it as indicated by the '/' to produce DNA products with 5' two-base overhangs. Owing to the nature of its cleavage pattern, it is likely that MspI would represent a new structural class of restriction endonucleases. Crystals of the dimeric MspI restriction enzyme bound to a duplex DNA molecule containing the specific recognition sequence have been obtained by vapor-diffusion techniques in the presence of polyethylene glycol as precipitant. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 50.2, b = 131.6, c = 59.3 A, beta = 109.7 degrees. The crystals contain one dimeric complex in the asymmetric unit. A complete native data set has been collected to a resolution of 2.05 A by cryo-crystallographic methods, with an R(merge) of 4.0%.

Crystallization↗

PubMed2005

Functional comparison of single- and double-stranded mdr1 antisense oligodeoxynucleotides in human ovarian cancer cell lines.

PURPOSE: P-glycoprotein mediated multidrug resistance presents a major obstacle in the successful therapeutic treatment of solid tumors such as ovarian cancer. Among the more promising techniques used to overcome multidrug resistance in ovarian cancer, is the transcriptional suppression of P-glycoprotein by antisense oligodeoxynucleotides (ODNs). To design more potent antisense ODNs, we explored the concept that double-stranded antisense ODNs may offer advantages in stability and potency over single-stranded in analogy to double-stranded siRNA. METHOD: Single-stranded phosphorothioate antisense ODNs against the human mdr1 gene were compared to the duplex of the active antisense and sense sequence of the same length. Concentration dependant effects on P-glycoprotein (Pgp) expression and functionality were quantitatively compared in the Pgp overexpressing ovarian cancer cell line A2780/Adr and its parental cell line A2780. Antisense ODNs were (111)Indium- and fluorescein isothiocyanate-conjugated for stability, cellular uptake and nuclear localization studies. Duplex formation significantly enhanced transcriptional inhibition of Pgp surface expression and functionality. Cellular uptake and distribution to the nucleus was improved when utilized as double-stranded DNA. CONCLUSION: Novel findings from this study suggest that double-stranded antisense ODNs more effectively inhibit target protein expression and consequently enhance chemoresponsiveness through improvements in cellular uptake and distribution to the nucleus.

Cell Line, Tumor↗

PubMed1991

Phased adenine tracts in double-stranded RNA do not induce sequence-directed bending.

Tracts of four to six adenines phased with the DNA helix produce a sequence-directed bending of the helix axis. Here, using gel electrophoresis and electron microscopy (EM), we have asked whether a similar motif will induce bending in a duplex RNA helix. Single-stranded RNAs were transcribed either from short synthetic DNA templates or from Crithidia fasciculata kinetoplast bent DNA, and the complementary single-stranded RNAs were annealed to produce duplex RNA molecules containing blocks of four to six adenines. Electrophoresis on polyacrylamide gels revealed no retardation of the RNAs containing phased blocks of adenines relative to duplex RNAs lacking such blocks. Examination by EM showed most of the molecules to be straight or only slightly bent. Thus, in contrast to DNA duplexes, phased adenine tracts do not induce sequence-directed bending in double-stranded RNA. Analysis of the distribution of molecule shapes for the highly bent C. fasciculata DNA showed that the adenine blocks do not act cooperatively to induce DNA bending and that the molecules must equilibrate between a spectrum of bent shapes.

Animals↗

PubMed2000

Discriminating duplex and hairpin oligonucleotides using chemical shifts: application to the anticodon stem-loop of Escherichia coli tRNA(Phe).

A sensitive NMR spectroscopic method for detection of duplex forms of self-complementary nucleic acid sequences has been implemented. The G.U wobble base pair formed between a (15)N-labeled strand and an unlabeled probe strand is used to identify the duplex. The guanine imino resonance, with its characteristic chemical shift, is detected using a 2D (15)N-(1)H heteronuclear multiple quantum coherence (HMQC) spectrum and provides a sensitive and unambiguous route to hairpin-duplex discrimination. The method has been used to identify the duplex and hairpin forms of an RNA oligonucleotide at concentrations of approximately 20 microM. This method has also been used to rule out possible duplex formation of an RNA oligonucleotide corresponding to the unmodified anticodon stem-loop of Escherichia coli tRNA(Phe) and suggests that this hairpin has a 3 nt loop.

Anticodon↗

PubMed1984

Breakage of single-stranded DNA by eukaryotic type 1 topoisomerase occurs only at regions with the potential for base-pairing.

Eukaryotic type 1 DNA topoisomerases break single-stranded DNA at specific sites. A preferred site for rat liver topoisomerase breakage in single-stranded phi X174 DNA was located within a region of the DNA with the potential for duplex formation. To investigate the relationship between sites of breakage in duplex and single-stranded DNA, a restriction fragment containing sequences from the transcriptional regulatory and enhancer region of the simian virus 40 genome was used as a substrate for topoisomerase. While different patterns of breakage in the native and denatured forms of the DNA were observed, some sites of breakage were common to both forms. The break sites in the denatured DNA were a subset of the break sites in the duplex DNA and were located in regions which had the potential for intrastrand base-pairing due to distal complementary sequences. A series of single-stranded fragments were generated with the distal complementary sequences deleted and these fragments were used as substrates for topoisomerase breakage. The lack of detectable breakage at a site when the complementary sequence was deleted, suggests that topoisomerase acts at duplex regions in the single-stranded DNA and that it is not active on regions of single-stranded DNA that are not base-paired.

Animals↗