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PubMed1992

Identification of genes using oligonucleotides corresponding to splice site consensus sequences.

The identification of genes in genomic DNA presents challenging technical difficulties. We show here the feasibility of using short oligonucleotides based on the consensus sequences surrounding intron-exon junctions to detect random phage and cosmid clones containing genes both through the analysis of DNA blots and by direct screening. Three degenerate oligonucleotides, a 10-mer corresponding to the 5' splice junction and a 9-mer and a 15-mer corresponding to the 3' splice junction, were tested on the known intron-exon boundaries of the cloned human proteolipid protein (PLP) gene at hybridization and washing temperatures appropriate to their length and composition. All predicted hybridizations were observed. The oligonucleotides were also used to identify random genomic plasmid and cosmid clones containing putative intron-exon junctions; the presence of genes in these clones was supported by RNA blot analysis and by cross-hybridization to DNA from other species. This technique should facilitate the identification of genes for inherited diseases by positional cloning studies and will assist in the identification of genes in random clones for the human genome project.

Base Sequence↗
PubMed1990

Complexity charts can be used to map functional domains in DNA.

We measured local compositional complexity (LCC) of DNA sequences by calculating Shannon information content over mononucleotide frequencies. Eukaryotic DNA appeared to be "simpler" than bacterial DNA even at the level of short oligonucleotides. Moreover, different DNA functional domains displayed different compositional complexity in a systematic manner. In particular, the complexity of exon sequences was systematically higher than the complexity of corresponding introns. We therefore present examples of complexity charts (plots of complexity versus position in sequence) for pre-mRNA sequences from higher eukaryotes. By taking a window width of 100 nucleotides and a window step of 1 nucleotide, introns can be distinguished from exons in the majority of cases studied. Complexity charts of immunoglobulin variable regions allowed correct mapping of exons and introns in these sequences as well, a task that was impossible with commercial programs available to date.

Acanthamoeba↗
PubMed2004

Pharmacokinetics and biodistribution of novel aptamer compositions.

PURPOSE: Aptamers are highly selective nucleic acid-based drugs that are currently being developed for numerous therapeutic indications. Here, we determine plasma pharmacokinetics and tissue distribution in rat of several novel aptamer compositions, including fully 2'-O-methylated oligonucleotides and conjugates bearing high-molecular weight polyethylene glycol (PEG) polymers, cell-permeating peptides, and cholesterol. METHODS: Levels of aptamer conjugates in biological samples were quantified radiometrically and by a hybridization-based dual probe capture assay with enzyme-linked fluorescent readout. Intact aptamer in urine was detected by capillary gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF). RESULTS: Aptamer compositions examined exhibited a wide range of mean residence times in circulation (0.6-16 h) and significant variation in distribution levels among organs and tissues. Among the conjugates tested, in vivo properties of aptamers were altered most profoundly by conjugation with PEG groups. Complexation with a 20 kDa PEG polymer proved nearly as effective as a 40 kDa PEG polymer in preventing renal clearance of aptamers. Conjugation with 20 kDa PEG prolonged aptamer circulatory half-life, while reducing both the extent of aptamer distribution to the kidneys and the rate of urinary elimination. In contrast, the fully 2'-O-Me aptamer composition showed rapid clearance from circulation, and elimination with intact aptamer detectable in urine at 48 h post-administration. CONCLUSIONS: We find that conjugation and chemical composition can alter fundamental aspects of aptamer residence in circulation and distribution to tissues. Though the primary effect of PEGylation was on aptamer clearance, the prolonged systemic exposure afforded by presence of the 20 kDa moiety appeared to facilitate distribution of aptamer to tissues, particularly those of highly perfused organs.

Animals↗
PubMed1999

Human Rad51 protein can form homologous joints in the absence of net strand exchange.

The eukaryotic homologs of RecA protein are central enzymes of recombination and repair, and notwithstanding a high degree of conservation they differ sufficiently from RecA to offer insights into mechanisms and biological roles. The yield of DNA strand exchange reactions driven by both Escherichia coli RecA protein and its human homolog HsRad51 protein was inversely related to the GC content of oligonucleotide substrates, but at any given GC composition, HsRad51 promoted less exchange than RecA. When 40% of bases were GC pairs, the rate constant for strand exchange by HsRad51 was unmeasurable, whereas the rate constants for homologous pairing were unaltered relative to more AT-rich DNA. The ability of HsRad51 to form joints in the absence of net strand exchange was confirmed by experiments in which heterologous blocks at both ends of linear duplex oligonucleotides produced joints that instantly dissociated upon deproteinization. These findings suggest that HsRad51 acting alone on human DNA in vivo is a pairing protein that cannot form extensive heteroduplex DNA.

Base Pair Mismatch↗
PubMed1992

Cloning and primary structure of neurocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain.

We have obtained the complete coding sequence of neurocan, a chondroitin sulfate proteoglycan of rat brain which is developmentally regulated with respect to its molecular size, concentration, carbohydrate composition, sulfation, and immunocytochemical localization. Two degenerate oligonucleotides, based on amino acid sequence data from the proteoglycan isolated from adult brain by immunoaffinity chromatography with the 1D1 monoclonal antibody, were used as sense and antisense primers in the polymerase chain reaction with a brain cDNA library as template to generate an unambiguous cDNA probe. A second probe for the N-terminal portion of the early postnatal form of the proteoglycan was obtained by reverse transcription/polymerase chain reaction. The composite sequence of overlapping cDNA clones is 5.2-kilobases (kb) long, including 1.3 kb of 3'-untranslated sequence and 76 base pairs of 5'-untranslated sequence. An open reading frame of 1257 amino acids encodes a protein with a molecular mass of 136 kDa containing 10 peptide sequences present in the adult and/or early postnatal brain proteoglycans. The deduced amino acid sequence revealed a 22-amino acid signal peptide followed by an immunoglobulin domain, tandem repeats characteristic of the hyaluronic acid-binding region of aggregating proteoglycans, and an RGDS sequence. The C-terminal portion (amino acids 951-1215) has approximately 60% identity to regions in the C termini of the fibroblast and cartilage proteoglycans, versican and aggrecan, including two epidermal growth factor-like domains, a lectin-like domain, and a complement regulatory protein-like sequence. The central 595-amino acid portion of neurocan has no homology with other reported protein sequences. The proteoglycan contains six potential N-glycosylation sites and 25 potential threonine O-glycosylation sites. In the adult form of the proteoglycan (which represents the C-terminal half of neurocan) a single 32-kDa chondroitin 4-sulfate chain is linked at serin-944, whereas three additional potential chondroitin sulfate attachment sites (only two of which are utilized) are present in the larger proteoglycan species. A probe corresponding to a region of neurocan having no homology with versican or aggrecan hybridized with a single band at approximately 7.5 kb on Northern blots of mRNA from both 4-day and adult rat brain (but not with muscle, kidney, liver, or lung mRNA), indicating that the 1D1 proteoglycan of adult brain, containing a 68-kDa core protein, is generated by a developmentally regulated in vivo proteolytic processing of the 136-kDa species which is predominant in early postnatal brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Aggrecans↗
PubMed2003

Expanding the design horizon of antisense oligonucleotides with alpha-L-LNA.

Oligonucleotides containing Locked Nucleic Acids (LNA) to various extents and at various positions were evaluated for antisense activity, RNase H recruitment, nuclease stability and thermal affinity. In this work, two different diastereoisomers of LNA were studied: the beta-D-LNA and the alpha-L-LNA (abbreviated as beta-D-LNA and alpha-L-LNA). Our findings show that the best antisense activity with 16mer gapmers containing beta-D-LNA (oligonucleotides containing consecutive segments of LNA and DNA with a central DNA stretch flanked by two LNA segments, LNA-DNA-LNA) is found with gap sizes between 7 and 10 nt. The optimal gap size is motif-dependent, and requires the right balance between gap size and affinity. Compared to beta-D-LNA, alpha-L-LNA shows superior stability against a 3'-exonuclease. The design possibilities of alpha-L-LNA were explored for different gapmers and other designs, collectively called chimeras. The placement of alpha-L-LNA in the junctions or in the flanks resulted in potent antisense oligonucleotides. Moreover, different chimeras with an alternate composition of DNA, alpha-L-LNA and beta-D-LNA were evaluated in terms of antisense activity and RNase H recruitment. Chimeras with an interrupted DNA stretch with alpha-L-LNA still recruit RNase H and show good levels of antisense activity, while the same design with beta-D-LNA results in a drop in antisense potency. Our findings indicate that alpha-L-LNA is a powerful and versatile nucleotide analogue for designing potent antisense oligonucleotides.

Base Sequence↗
PubMed2005

Liposome-oligonucleotides interaction for in vitro uptake by COS I and HaCaT cells.

Liposomes are considered very promising delivery systems for antisense therapeutic approach, offering drug protection and facilitating oligonucleotide cell internalization. The present study was aimed to investigate the influence of phospholipid composition of the liposomal systems both on the encapsulation and on the oligonucleotide carrier capacity in vitro. Liposomes composed of neutral (phosphatidylcholine, cholesterol and dioleoylphosphatidylethanolamine) and/or cationic lipids (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride salt, DOTAP) with different molar ratios were complexed with 5' fluorescein conjugated 29-mer phosphorothioate oligonucleotide (PS-ODN). The interaction was evaluated using atomic force microscopy (AFM), gel electrophoresis and HPLC analysis. Cytofluorimetric analysis and fluorescence microscopy were applied to evaluate the uptake and intracellular distribution of fluorescently labelled PS-ODN after transfection in two cell lines, COS I (fibroblast cell) and HaCaT (immortalized keratinocyte cell). The AFM studies reveal that the liposome/PS-ODN interaction leads the formation of a new irregular structure that completely hides the PS-ODN. Gel electrophoresis experiments and HPLC analysis have clearly demonstrated that also neutral liposomes are able to keep a little amount of PS-ODN but without strain to the complexation; the interaction was weak and rapidly destabilized when the complex was added to the cells. Transfection experiments performed with different incubation times show that DOTAP liposomes increase the rate of cellular uptake of PS-ODN and seem to influence its intracellular distribution in COS I cells where the oligonucleotide looks localized in nucleoli. Similar behaviour, at a lesser extent, is exhibited in HaCaT cells.

Animals↗
PubMed2003

Size and composition of T-cell receptor delta (TCRD) junctional sequences are not predictive of the sensitivity of clonospecific oligonucleotides designed for detection of minimal residual disease in acute lymphoblastic leukemia.

We characterized 168 junctional regions of T-cell receptor delta (TCRD) rearrangements from 116 children with acute lymphoblastic leukemia (ALL) (101 with precursor B-cell ALL, 15 with T-cell ALL). Application of 101 allele-specific oligonucleotide (ASO) probes representing 85 Vdelta2Ddelta3, 10 Ddelta2Ddelta3, 3 Vdelta1Jdelta1, 1 Vdelta3Jdelta1, and 2 Ddelta2Jdelta1 junctions for the detection of minimal residual disease (MRD) revealed detection levels of 10(-4) to 10(-6) leukemia cells in the vast majority of cases (93 of 101). Of interest was that neither the N, D, P (nontemplated, diversity, palindromic) content and length of the junctional regions nor the number of nucleotides deleted from the flanking V, D, or J (variable, diversity, joining) elements correlated with the sensitivity of ASO probes. These data indicated that in ALL TCRD rearrangements can serve as suitable tools for the detection of MRD irrespective of the specific composition of the junctional region.

Base Sequence↗
PubMed1979

DNA chain length markers and the influence of base composition on electrophoretic mobility of oligodeoxyribonucleotides in polyacrylamide-gels.

The specific influence of the four nucleobases on electrophoretic mobility of oligodeoxyribonucleotides in polyacrylamide-gels under denaturing and nondenaturing conditions has been investigated using homooligomers from the four deoxyribonucleotides as chain length standards. Homooligomers of same chain lengths exhibit remarkable differences in mobility. Specific retardation of any other oligonucleotide investigated was found to be mainly dependent on base composition but not on sequence. A simple procedure is presented for calculating mobilities relative to the standards on denaturing gels. This allows a reliable identification of oligonucleotides on acrylamide-gels by exact chain length determination with respect to base composition and furthermore a detailed interpretation of complex reaction mixtures. The homooligomers also show the same differences in mobility on nondenaturing gels. The significance of this effect for strand separation is discussed.

Base Sequence↗
PubMed1998

Transition from SCLC to NSCLC phenotype is accompanied by an increased TRE-binding activity and recruitment of specific AP-1 proteins.

Transitions from small cell (SCLC) to non-small cell lung cancer (NSCLC) cells have been documented both in vitro and in vivo and are thought to be an important step during tumor progression of human small cell lung cancer towards a treatment-resistant tumor state. We have screened NSCLC and SCLC cell lines for differences in the composition of nuclear transcription factors using consensus oligonucleotide sequences (SRE, Ets, TRE, CRE, B-motif, GAS, E-box). We found NSCLC cells to exhibit significantly higher AP-1 binding activity than SCLC cells consistent with the increased expression of CD44, an AP-1 target gene. To gain more insight into the molecular mechanisms underlying these differences, we analysed SCLC cell lines (NCI-N592 and NCI-H69) which were phenotypically transformed into NSCLC-type cells by transfection with activated H-ras and c-myc oncogenes. In these cells, ras-induced transition is accompanied by a strong induction of AP-1-binding activity along with increased expression of CD44 mRNA and protein. When analysing the composition of the AP-1 complex in more detail and comparing ras-induced versus phorbol ester-induced changes, we found Fra-1 to be the major component induced in ras-transfected but not in phorbol-ester treated or non-treated parental SCLC cells. This finding is paralleled by the observation that among the various members of the Fos and Jun family analysed (c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunD, JunB) fra-1 is the only gene to be exclusively expressed in NSCLC cells but not in cells of SCLC origin. Our data, thus, point to a histiotype-related mechanism of recruitment among AP-1 proteins which may have bearings on the fate of lung cancer development.

Carcinoma, Non-Small-Cell Lung↗
PubMed1992

Triple-helix formation is compatible with an adjacent DNA-protein complex.

The effect of oligonucleotide-directed triple-helix formation on the binding of a protein to an immediately adjacent sequence has been examined. A double-stranded oligonucleotide was designed with a target site for the binding of a pyrimidine oligonucleotide located immediately adjacent to the recognition sequence for the herpes simplex virus type 1 (HSV-1) origin of replication binding protein, which is encoded by the UL9 gene of HSV-1. Since the optimal conditions for the binding of the UL9 protein and the pyrimidine oligonucleotide to the duplex DNA are markedly different, a pyrimidine oligonucleotide was designed to optimize binding affinity and specificity for the target duplex oligonucleotide. Consideration was given to length and sequence composition in an effort to maximize triple-strand formation under conditions amenable to the formation of the UL9-DNA complex. Using gel mobility shift assays, a trimolecular complex composed of duplex DNA bound to both a third oligonucleotide strand and the UL9 protein was detected, indicating that the UL9-DNA complex is compatible with the presence of a triple helix in the immediately adjacent sequences.

Animals↗
PubMed2000

Synthesis of 8-(hydroxymethyl)-3,N(4)-etheno-2'-deoxycytidine, a potential carcinogenic glycidaldehyde adduct, and its site-specific incorporation into DNA oligonucleotides.

The previously unreported glycidaldehyde adduct, 8-(hydroxymethyl)-3, N(4)-etheno-2'-deoxycytidine (8-HM-epsilondC), has been synthesized for the first time by reaction of 2'-deoxycytidine with bromoacetaldehyde at pH 4.5, followed by reduction with sodium borohydride. The adduct was characterized by UV, MS, and NMR. The compound was stable to neutral and acidic conditions but not in alkaline solution. The corresponding phosphoramidite was synthesized in good yield from the intermediate, 3, N(4)-ethenocarbaldehyde-2'-deoxycytidine, using the standard methodology and site-specifically incorporated in both 15- and 25-mer oligonucleotides, for studies on biochemical and biophysical properties. The resulting oligonucleotides were purified using HPLC, and the base composition was verified by HPLC after enzymatic digestion.

Chromatography, High Pressure Liquid↗
PubMed1995

Noncovalent protein--oligonucleotide interactions monitored by matrix-assisted laser desorption/ionization mass spectrometry.

Positive ion mode matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to explore nonspecific interactions between proteins and oligonucleotides. The formation of noncovalent complexes showed correlation with the type of oligonucleotide bases and with the amino acid composition of the proteins. Among the four DNA homooligomers, abundant protein-nucleic acid complexes were detected for pd(T)n, whereas negligible attachment was evident for pd(A)n, pd(C)n, and pd(G)n. Mixed base sequence nucleic acids (pd(AGCTCAGCTT) and d(TTAGCAGCTT) also showed affinity to Arg-Lys. The protein affinity of pd(T)n turned out to be nonspecific and produced a larger variety of complexes when the number of basic residues in the protein was increased. Complexation of pd(T)n with small basic dipeptides (Arg-Lys or His-His) led to significant improvement in the mass resolution for positive ions. For example, the mass resolution of the pd(T)20/Arg-Lys complex exhibited about 4 times improvement over pd(T)20 alone. The protein--oligonucleotide interactions were also pH and matrix dependent. Lowering the pH from its original value (pH = 1.7) led to diminishing complex related signal, whereas increasing the pH resulted in the appearance of a larger variety of complexes. 2,5-Dihydroxybenzoic acid matrix demonstrated much greater tendency to produce complex ions than did the three other matrix materials we tested. A possible explanation of the observed phenomena was based on pH-controlled ion pair formation between oligonucleotides and proteins.

Acid-Base Equilibrium↗
PubMed2007

Nonspecific cellular accumulation of 99mTc-labeled oligonucleotides in culture is influenced by their guanine content.

Specific accumulation of radiolabeled antisense oligonucleotides as a result of binding by an antisense mechanism to target mRNAs in tumor has been repeatedly observed. However, the mechanisms responsible for nonspecific cellular accumulation remain almost completely unexplored. We have occasionally observed in cell culture nonspecific accumulations of 99mTc-labeled sense, scrambled or random control oligonucleotides in tumor cells comparable to or even higher than those of the corresponding antisense oligonucleotides. We have also observed that these nonspecific accumulations of control oligonucleotides are sequence dependent. To explore the influence of base composition on nonspecific accumulation, we used MCF-7 breast cancer cells, along with 10 uniform phosphorothioates and 5 uniform phosphodiesters oligonucleotides. Three of the phosphorothioates were antisense against different sites within the survivin mRNA, and two were the corresponding sense and scrambled controls. In addition, the accumulations in MCF-7 cells of radiolabeled poly A, poly C, poly T and poly GGGA phosphorothioate oligonucleotides were also studied to explore the influence of each nitrogenous base on the nonspecific cell accumulations of phosphorothioate oligonucleotides. Our results show that guanine content is an important determinant of nonspecific cellular accumulations under the conditions of this investigation. If this observation can be shown to be universally applicable to other cell types, then the selection of control sequences in studies of antisense tumor targeting should avoid those that are guanine rich, if possible.

Breast Neoplasms↗
PubMed1975

Complementary binding of oligonucleotides with 16S RNA and ribosomal ribonucleoproteins.

The accessibility of single-stranded sequences in 16S RNA in free state and in ribonucleoprotein particles (RNP) to complementary binding with isoplith fractions of oligonucleotides was studied. RNP had different protein composition and corresponded to intermediate stages of E. coli 30S subunit assembly in vitro. Gel-filtration was used to detect the most strong binding. It was found that S4 essentially inhibited the hexamer binding to RNA. 'Core' proteins bound to 16S RNA strongly increased the shielding of single-stranded regions while 'split' proteins insignificantly changed the hexamer binding. Nevertheless evidence is presented that 'split' proteins might also interact directly with 16S RNA in the 30S subunit.

Bacterial Proteins↗
PubMed1982

Analysis of vaccinia virus transcriptional complexity in vitro and in vivo: characterization of RNase T1-resistant 5'-terminal oligonucleotides.

Vaccinia virus mRNAs synthesized in vitro and in vivo, polyadenylated leader sequences synthesized in vitro in the absence of added GTP, CTP, or UTP or in the presence of 20 micrograms of actinomycin D per ml, and high-molecular-weight RNA synthesized in vitro under limiting ATP concentrations were labeled specifically in the cap structure using [alpha-32P]GTP and vaccinia-soluble enzyme extracts. The complexity of RNase T1-resistant 5'-terminal oligonucleotides was analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 190 unique T1-resistant 5'-terminal oligonucleotides were observed from vaccinia virus 8 to 12S RNA synthesized in vitro. A somewhat greater complexity was observed with polyadenylated leader sequences and actinomycin D RNAs where unique T1-resistant oligonucleotides ranged from approximately 210 to 280 5'-terminal fragments. On a composite fingerprint of the above RNAs, more than 300 identifiable unique T1-resistant 5'-terminal oligonucleotides were observed. Significantly, close to 300 T1-resistant fragments were derived from RNA sedimenting faster than 18S on denaturing sucrose gradients. Analysis of vaccinia RNAs synthesized in vivo in the absence of either de novo protein synthesis or DNA replication or in the presence of actinomycin D gave essentially similar profiles of 5'-terminal T1-resistant oligonucleotide fingerprints consisting of approximately 200 fragments. Analysis of the 5'-terminal T1-resistant oligonucleotides of vaccinia RNAs present after DNA replication showed essentially the same pattern of early T1-fragments albeit in reduced amounts but in addition revealed a complex pattern of T1-resistant oligonucleotides unique to this class of vaccinia RNA.

Adenosine Triphosphate↗
PubMed2005

Evaluation of custom-made DNA microarrays for multilocus sequence typing of Neisseria meningitidis.

Multilocus sequence typing (MLST) has become the gold standard for typing of a variety of bacterial and fungal micro-organisms. Others recently reported the successful use of the tiling DNA array technology to sequence-type Staphylococcus aureus. We now evaluated microarrays based on polymorphism-directed oligonucleotide design for typing of Neisseria meningitidis. The rationale behind this approach was to minimize the number of microarray probes by exploiting the comprehensive knowledge of polymorphisms combined in the Neisseria MLST website. Initial experiments using model oligonucleotides of 28-32 base-pairs in length revealed that the hybridization protocols used were highly specific. However, despite of several optimization steps, the rate of misidentification of oligonucleotides remained > 1.8% in consecutive validation experiments using arrays representing the genetic diversity at three MLST loci. We assume that the high density of polymorphic sites and the extensive GC-content variations at N. meningitidis MLST loci hinder the successful implementation of MLST microarrays based on polymorphism-directed oligonucleotide design.

Alleles↗