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PubMedDate not supplied

Antisense DNA and RNA strategies: new approaches to therapy.

This review describes the results both in vitro and in vivo of the application of small DNA molecules or their analogues to target sequences in messenger RNA (mRNA) or DNA. Biological effects on the replication of viruses and the expression of oncogenes are recorded. At the same time, RNA catalytic sequences ('ribozymes') have been used to target and cleave mRNA sequences. Before these treatments can be confidently applied to clinical situations, further work is needed on their stability, cellular uptake and selection of their target, and the mechanism of their action also needs to be understood and controlled. In this way, it may be possible to guarantee specific effects on gene or cell type. The review describes the current research and state of development.

Animals↗
PubMed1994

Homology dependence of targeted recombination at the Chinese hamster APRT locus.

Using simple linear fragments of the Chinese hamster adenine phosphoribosyltransferase (APRT) gene as targeting vectors, we have investigated the homology dependence of targeted recombination at the endogenous APRT locus in Chinese hamster ovary (CHO) cells. We have examined the effects of varying either the overall length of targeting sequence homology or the length of 5' or 3' flanking homology on both the frequency of targeted homologous recombination and the types of recombination events that are obtained. We find an exponential (logarithmic) relationship between length of APRT targeting homology and the frequency of targeted recombination at the CHO APRT locus, with the frequency of targeted recombination dependent upon both the overall length of targeting homology and the length of homology flanking each side of the target gene deletion. Although most of the APRT+ recombinants analyzed reflect simple targeted replacement or conversion of the target gene deletion, a significant fraction appear to have arisen by target gene-templated extension and correction of the targeting fragment sequences. APRT fragments with limited targeting homology flanking one side of the target gene deletion yield proportionately fewer target gene conversion events and proportionately more templated extension and vector correction events than do fragments with more substantial flanking homology.

Adenine Phosphoribosyltransferase↗
PubMed2003

Rhythmic movement is attracted more strongly to auditory than to visual rhythms.

People often move in synchrony with auditory rhythms (e.g., music), whereas synchronization of movement with purely visual rhythms is rare. In two experiments, this apparent attraction of movement to auditory rhythms was investigated by requiring participants to tap their index finger in synchrony with an isochronous auditory (tone) or visual (flashing light) target sequence while a distractor sequence was presented in the other modality at one of various phase relationships. The obtained asynchronies and their variability showed that auditory distractors strongly attracted participants' taps, whereas visual distractors had much weaker effects, if any. This asymmetry held regardless of the spatial congruence or relative salience of the stimuli in the two modalities. When different irregular timing patterns were imposed on target and distractor sequences, participants' taps tended to track the timing pattern of auditory distractor sequences when they were approximately in phase with visual target sequences, but not the reverse. These results confirm that rhythmic movement is more strongly attracted to auditory than to visual rhythms. To the extent that this is an innate proclivity, it may have been an important factor in the evolution of music.

Acoustic Stimulation↗
PubMed2004

Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction.

An efficient and effective method for quantification of small amounts of nucleic acids contained within a sample specimen would be an important diagnostic tool for determining the content of mitochondrial DNA (mtDNA) in situations where the depletion thereof may be a contributing factor to the exhibited pathology phenotype. This study compares two quantification assays for calculating the total mtDNA molecule number per nanogram of total genomic DNA isolated from human blood, through the amplification of a 613-bp region on the mtDNA molecule. In one case, the mtDNA copy number was calculated by standard competitive polymerase chain reaction (PCR) technique that involves co-amplification of target DNA with various dilutions of a nonhomologous internal competitor that has the same primer binding sites as the target sequence, and subsequent determination of an equivalence point of target and competitor concentrations. In the second method, the calculation of copy number involved extrapolation from the fluorescence versus copy number standard curve generated by real-time PCR using various dilutions of the target amplicon sequence. While the mtDNA copy number was comparable using the two methods (4.92 +/- 1.01 x 10(4) molecules/ng total genomic DNA using competitive PCR vs 4.90 +/- 0.84 x 10(4) molecules/ng total genomic DNA using real-time PCR), both inter- and intraexperimental variance were significantly lower using the real-time PCR analysis. On the basis of reproducibility, assay complexity, and overall efficiency, including the time requirement and number of PCR reactions necessary for the analysis of a single sample, we recommend the real-time PCR quantification method described here, as its versatility and effectiveness will undoubtedly be of great use in various kinds of research related to mitochondrial DNA damage- and depletion-associated disorders.

DNA, Mitochondrial↗
PubMed2005

Sequence preference in DNA binding: de novo designed helix-turn-helix metallopeptides recognize a family of DNA target sites.

The DNA-binding behavior and target sequences of two designed metallopeptides have been investigated with an iterative electrophoresis mobility shift assay followed by PCR amplification, and by circular dichroism spectroscopy. Peptides P3W and P5b were designed based on the structural similarity of the helix-turn-helix motif of homeodomains and the EF-hand motifs of calmodulin, as previously described for P3W. Like P3W, P5b binds both Eu(III) (K(d) = 12.6 +/- 1.9 microM) and Ca(II) (K(d) = 70 +/- 8 microM) with reasonable affinity. Binding selection from a library of randomized 8-mer DNA oligonucleotide sequences identified one target family for CaP5b [5'-pur-T-pur-G-(G/C)-3'], and two target sites for CaP3W [5'-(A/T)-G-G-G-(T/C)-3' and 5'-A-T-(G/T)-T-G-3']. Circular dichroism studies indicate that unlike EuP3W, EuP5b is poorly folded in the absence of DNA. In the presence of DNA containing target-binding sites for both peptides, both EuP3W and EuP5b increase in helical content, in the latter case significantly. These results suggest that EuP5b binding to target DNA involves an induced-fit mechanism. These small chimeric metallopeptides have been found to bind selectively to DNA targets, analogous to natural protein-DNA interactions. This corroborates our earlier conclusions (J. Am. Chem. Soc. 125:6656, 2003) that sequence-preferential DNA cleavage by Ce(IV)P3W was due to sequence recognition.

Base Sequence↗
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↗
PubMed1994

The basolateral targeting signal in the cytoplasmic domain of glycoprotein G from vesicular stomatitis virus resembles a variety of intracellular targeting motifs related by primary sequence but having diverse targeting activities.

Using systematic site-directed mutagenesis, the basolateral targeting signal in the cytoplasmic domain of glycoprotein G from vesicular stomatitis virus (VSV G) has been localized to an 11-amino acid sequence, which contains two essential residues and a third that makes a minor contribution. A tyrosine at position 19 of the 29-residue carboxyl-terminal cytoplasmic tail is the most important residue and cannot be replaced by other aromatic amino acids, while an isoleucine at position 22, 3 residues carboxyl-terminal to this tyrosine, is also critical but can be replaced by other aliphatic residues. Additionally, an arginine at position 16 makes a minor contribution. Therefore the crucial elements of this targeting signal can be represented by the sequence Y-X-X-aliphatic. While earlier investigation has suggested similarity between basolateral targeting and internalization signals, alignment of this sequence with other cytoplasmic targeting signals suggests the existence of a broad class of homologous targeting motifs that direct protein delivery to a variety of cellular locations. This in turn suggests the existence of a family of homologous receptors, distributed throughout the cell, which differ in their affinity for subsets of these targeting sequences.

Amino Acid Sequence↗
PubMed2004

Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNA.

The bacterial transposon Tn10 inserts preferentially into specific target sequences. This insertion specificity appears to be linked to the ability of target sites to adopt symmetrically positioned DNA bends after binding the transposition machinery. Target DNA bending is thought to permit the transposase protein to make additional contacts with the target DNA, thereby stabilizing the target complex so that the joining of transposon and target DNA sequences can occur efficiently. In the current work, we have asked whether the introduction of a discontinuity in a target DNA strand, a modification that is expected to make it easier for a DNA molecule to bend, can enhance or rescue target capture under otherwise suboptimal reaction conditions. We show that either a nick or a missing phosphate specifically at the site of reaction chemistry increases the ability of various target DNAs to form the target capture complex. The result suggests that the bends in the target DNA are highly localized and include the scissile phosphates. This raises the possibility that strand transfer is mechanistically linked to target capture. We have also identified specific residues in the target DNA and in transposase that appear to play an important role in target DNA bending.

Base Sequence↗
PubMed1999

Triple helix formation and the antigene strategy for sequence-specific control of gene expression.

Specific gene expression involves the binding of natural ligands to the DNA base pairs. Among the compounds rationally designed for artificial regulation of gene expression, oligonucleotides can bind with a high specificity of recognition to the major groove of double helical DNA by forming Hoogsteen type bonds with purine bases of the Watson-Crick base pairs, resulting in triple helix formation. Although the potential target sequences were originally restricted to polypurine-polypyrimidine sequences, considerable efforts were devoted to the extension of the repertoire by rational conception of appropriate derivatives. Efficient tools based on triple helices were developed for various biochemical applications such as the development of highly specific artificial nucleases. The antigene strategy remains one of the most fascinating fields of triplex application to selectively control gene expression. Targeting of genomic sequences is now proved to be a valuable concept on a still limited number of studies; local mutagenesis is in this respect an interesting application of triplex-forming oligonucleotides on cell cultures. Oligonucleotide penetration and compartmentalization in cells, stability to intracellular nucleases, accessibility of the target sequences in the chromatin context, the residence time on the specific target are all limiting steps that require further optimization. The existence and the role of three-stranded DNA in vivo, its interaction with intracellular proteins is worth investigating, especially relative to the regulation of gene transcription, recombination and repair processes.

Animals↗
PubMed2004

Characterization and quantification of triple helix formation in chromosomal DNA.

DNA-binding molecules that recognize specific sequences offer a high potential for the understanding of chromatin structure and associated biological processes in addition to their therapeutic potential, e.g. as positioning agents for validated anticancer drugs. A prerequisite for the development of DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the human genome. We have further developed a capture assay to assess triplex-forming oligonucleotide (TFO) binding efficiency quantitatively. This assay is based on bifunctional, psoralen and biotin-conjugated, TFOs and real-time PCR analysis. We have applied this novel quantification method to address two issues that are relevant for DNA-binding molecules. First, we have compared directly the extent of TFO-binding in three experimental settings with increasing similarity to the situation in vivo, i.e. naked genomic DNA, isolated cell nuclei, or whole cells. This comparison allows us to characterize factors that influence genomic triplex formation, e.g. chromosomal DNA organization or intracellular milieu. In isolated nuclei, the binding was threefold lower compared to naked DNA, consistent with a decreased target accessibility int he nucleosomal environment. Binding was detected in whole cells, indicating that the TFO enters the nucleus and binds to its target in intact cells in vivo, but the efficiency was decreased (tenfold) compared to nuclei. Secondly, we applied the method to characterize the binding properties of two different TFOs targeting the same sequence. We found that an antiparallel-binding GT-containing TFO bound more efficiently, but with less target sequence selectivity compared to a parallel-binding CU-containing TFO. Collectively, a sensitive method to characterize genomic triplex formation was described. This may be useful for the determination of factors driving TFO binding efficiency and, thus, may improve the usefulness of triplex-mediated gene targeting for studies of chromatin structure as well as for therapeutic antigene strategies.

Base Sequence↗
PubMed1993

A PCR-derived library of random point mutations within the V3 region of simian immunodeficiency virus.

Oligonucleotide primers corresponding to variable region 3 (V3) of simian immunodeficiency virus (SIV) were randomly mutagenized during synthesis by doping each of the four nucleoside phosphoramidites with a small amount of the other three. PCR was then used to incorporate the altered sequences into larger, clonable DNA fragments by spliced overlap extension (SOE). With the composition of the phosphoramidites used, 53 of the 100 clones analyzed were unique, having one or more point mutation within the 84-bp target sequence. These 53 unique clones contained an average of 2.1 nucleotide substitutions and 1.5 amino acid substitutions per clone within the target V3 sequence. Of the internal 25 amino acid positions within the V3 domain, 23 were changed at least once. This method should be generally useful for the construction of libraries of random point mutations within a defined target DNA sequence.

Amino Acid Sequence↗
PubMed1996

Peptide nucleic acids directed to the promoter of the alpha-chain of the interleukin-2 receptor.

Two 10-mer oligopyrimidine peptide nucleic acids (PNAs) were designed to interfere with IL-2R alpha promoter expression by binding to the regulatory sequences overlapping SRF and NF-kappa B transcription factor sites. Specific complexes were formed on each target sequence, and clearly involved (1) Hoogsteen hydrogen bonds as shown by experiments in which the purine strand of a single or double-stranded target was substituted with 7-deazadeoxyguanosine, (2) P-loop formation on double-helical DNA as evidenced by susceptibility to a single-strand-specific nuclease. When formed on a single-stranded DNA target, these highly stable complexes were responsible for efficient physical blockage of T7 DNA polymerase elongation on the template DNA containing the target oligopurine sequence. On a double-stranded target, these complexes only formed at low ionic strength and were slowly dissociated at physiological ionic strength (pH 6.5) with a t1/2 of 6.5-7 h. The salt-dependent instability of preformed complexes on a plasmid target was probably the critical factor responsible for their lack of significant sequence-specific effect on IL-2R alpha promoter activity inside living cells.

Binding Sites↗
PubMed1987

Profile analysis: detection of distantly related proteins.

Profile analysis is a method for detecting distantly related proteins by sequence comparison. The basis for comparison is not only the customary Dayhoff mutational-distance matrix but also the results of structural studies and information implicit in the alignments of the sequences of families of similar proteins. This information is expressed in a position-specific scoring table (profile), which is created from a group of sequences previously aligned by structural or sequence similarity. The similarity of any other sequence (target) to the group of aligned sequences (probe) can be tested by comparing the target to the profile using dynamic programming algorithms. The profile method differs in two major respects from methods of sequence comparison in common use: (i) Any number of known sequences can be used to construct the profile, allowing more information to be used in the testing of the target than is possible with pairwise alignment methods. (ii) The profile includes the penalties for insertion or deletion at each position, which allow one to include the probe secondary structure in the testing scheme. Tests with globin and immunoglobulin sequences show that profile analysis can distinguish all members of these families from all other sequences in a database containing 3800 protein sequences.

Amino Acid Sequence↗
PubMed1996

Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition.

Human L1 elements are highly abundant poly(A) (non-LTR) retrotransposons whose second open reading frame (ORF2) encodes a reverse transcriptase (RT). We have identified an endonuclease (EN) domain at the L1 ORF2 N-terminus that is highly conserved among poly(A) retrotransposons and resembles the apurinic/apyrimidinic (AP) endonucleases. Purified L1 EN protein (L1 ENp) makes 5'-PO4, 3'-OH nicks in supercoiled plasmids, shows no preference for AP sites, and preferentially cleaves sequences resembling L1 in vivo target sequences. Mutations in conserved amino acid residues of L1 EN abolish its nicking activity and eliminate L1 retrotransposition. We propose that L1 EN cleaves the target site for L1 insertion and primes reverse transcription.

Amino Acid Sequence↗
PubMed2004

Characterization of cis-acting prgQ mutants: evidence for two distinct repression mechanisms by Qa RNA and PrgX protein in pheromone-inducible enterococcal plasmid pCF10.

The pCF10-encoded negative regulators PrgX and Qa (prgQ antisense) RNA inhibit pCF10 transfer by blocking prgQ transcription extension past a potential transcription terminator sequence IRS1. To identify potential target sites for negative regulation, we isolated and analysed 13 cis-acting mutations in the prgXQ region. Determination of the 3' end of Qa RNA showed that eight mutations mapped in the region encoding Qa RNA. Four mutations were in the Qa promoter region and one was in IRS1. Three mutations in Qa greatly reduced the intracellular level of this RNA but did not affect that of PrgX. However, both Qa RNA and PrgX protein were reduced in three Qa promoter region mutants and the expression of prgQ transcripts extending 3' from IRS1 became constitutive. Qa RNA could mediate its negative regulatory activity in the absence of PrgX, and this activity was not abolished by cCF10, the peptide pheromone that induces pCF10 transfer. RNA analysis showed that Qa RNA abolished transcription readthrough. Based on the experimental data as well as computer analysis of predicted secondary structures of prgQ mRNA in the presence or absence of Qa, we concluded that Qa RNA is a pheromone-insensitive effector of prgQ mRNA termination or degradation at IRS1. In cells lacking a Qa target sequence, expression of PrgX repressed transcription from the prgQ promoter, and this repression was relieved by addition of exogenous cCF10. Thus, even though the synthesis of these negative regulators is coupled, they each act independently on separate targets to regulate expression of conjugation functions.

Bacterial Proteins↗
PubMed1995

Inhibition of interleukin-2/p55 receptor subunit interaction by complementary peptides.

Complementary peptides to interleukin-2 (IL-2) sequences important for receptor binding were tested for their ability to mimic natural receptors and act as inhibitors of the IL-2/p55 receptor subunit interaction. Peptides hydropathically complementary to IL-2 sequences 15-27 and 40-54 were synthesized in a linear and in a multimeric form and then characterized first by solid-phase binding assays for their ability to interact with IL-2. Binding between the multimeric complementary peptides and biotinylated IL-2 was specific, saturable, and inhibited by linear as well as multimeric complementary peptides. Saturable interactions, characterized by dissociation constants in the micromolar range, occurred also between IL-2 immobilized on microtiter plates and biotinylated linear and multimeric complementary peptides. Peptides corresponding to the IL-2 target sequences were able to interfere with this interaction, as well as full-length IL-2. Peptide recognition was sequence dependent, since scrambling of complementary peptide sequences or IL-2 target peptide sequences abolished binding. Multimeric complementary peptides after immobilization on solid supports proved useful also for affinity purifications of recombinant IL-2 or IL-2 fragments corresponding to the target sites, directly from crude mixtures, in high yield and with high recovery. Complementary peptides to IL-2 sequence 15-27, but not to IL-2 sequence 40-54, in the linear or in the multimeric form, even if with different potency, interfered with the IL-2/p55 receptor subunit interaction in vitro, thus suggesting a possible role of this IL-2 site in receptor recognition.

Amino Acid Sequence↗
PubMed2001

Illusory conjunctions of pitch and duration in unfamiliar tone sequences.

In 3 experiments, the authors examined short-term memory for pitch and duration in unfamiliar tone sequences. Participants were presented a target sequence consisting of 2 tones (Experiment 1) or 7 tones (Experiments 2 and 3) and then a probe tone. Participants indicated whether the probe tone matched 1 of the target tones in both pitch and duration. Error rates were relatively low if the probe tone matched 1 of the target tones or if it differed from target tones in pitch, duration, or both. Error rates were remarkably high, however, if the probe tone combined the pitch of 1 target tone with the duration of a different target tone. The results suggest that illusory conjunctions of these dimensions frequently occur. A mathematical model is presented that accounts for the relative contribution of pitch errors, duration errors, and illusory conjunctions of pitch and duration.

Adolescent↗
PubMed1997

Sensitized photomodification of single-stranded DNA by a binary system of oligonucleotide conjugates.

A photoactivatable binary system of oligonucleotide conjugates that form reactive species when assembling on a target nucleotide sequence has been developed. The binary system consists of two oligonucleotides. One contains a photosensitizing group, and the second contains a photoreactive group. Binding of the oligonucleotides to adjacent sequences in the target nucleic acid brings the groups in contact, which allows transfer of the absorbed energy from the sensitizer to the reagent and triggers crosslinking of the reagent to the target. One advantage of the binary system is the improved specificity, which is determined by independent binding of two oligonucleotides to the target sequence. Another advantage is the very high efficiency of the reaction achieved because each molecule of the target-bound sensitizing conjugate can activate many photoreactive oligonucleotide conjugate molecules bound to the target sequence.

Bacteriophage M13↗