Coiled bodies contain U7 small nuclear RNA and associate with specific DNA sequences in interphase human cells.

Coiled bodies (CBs) are nuclear organelles whose structures appear to be highly conserved in evolution. In rapidly cycling cells, they are typically located in the nucleoplasm but are often found in contact with the nucleolus. The CBs in human cells contain a unique protein, called p80-coilin. Studies on amphibian oocyte nuclei have revealed a protein within the "sphere" organelle that shares significant structural similarity to p80-coilin. Spheres and CBs are also highly enriched in small nuclear ribonucleoproteins and other RNA-processing components. We present evidence that, like spheres, CBs contain U7 small nuclear RNA (snRNA) and associate with specific chromosomal loci. Using biotinylated 2'-O-methyl oligonucleotides complementary to the 5' end of U7 snRNA and fluorescence in situ hybridization, we show that U7 is distributed throughout the nucleoplasm, excluding nucleoli, and is concentrated in CBs. Interestingly, we found that CBs often associate with subsets of the histone, U1, and U2 snRNA gene loci in interphase HeLa-ATCC and HEp-2 monolayer cells. However, in a strain of suspension-grown HeLa cells, called HeLa-JS1000, we found a much lower rate of association between CBs and snRNA genes. Possible roles for CBs in the metabolism of these various histone and snRNAs are discussed.

Gene disruption of a G4-DNA-dependent nuclease in yeast leads to cellular senescence and telomere shortening.

The yeast gene KEM1 (also named SEP1/DST2/XRN1/RAR5) produces a G4-DNA-dependent nuclease that binds to G4 tetraplex DNA structure and cuts in a single-stranded region 5' to the G4 structure. G4-DNA generated from yeast telomeric oligonucleotides competitively inhibits the cleavage reaction, suggesting that this enzyme may interact with yeast telomeres in vivo. Homozygous deletions of the KEM1 gene in yeast block meiosis at the pachytene stage, which is consistent with the hypothesis that G4 tetraplex DNA may be involved in homologous chromosome pairing during meiosis. We conjectured that the mitotic defects of kem1/sep1 mutant cells, such as a higher chromosome loss rate, are also due to failure in processing G4-DNA, especially at telomeres. Here we report two phenotypes associated with a kem1-null allele, cellular senescence and telomere shortening, that provide genetic evidence that G4 tetraplex DNA may play a role in telomere functioning. In addition, our results reveal that chromosome ends in the same cells behave differently in a fashion dependent on the KEM1 gene product.

The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver.

Glycoproteins expressing the Lutheran blood group antigens were isolated from human erythrocyte membranes and from human fetal liver. Amino acid sequence analyses allowed the design of redundant oligonucleotides that were used to generate a 459-bp, sequence-specific probe by PCR. A cDNA clone of 2400 bp was isolated from a human placental lambda gt 11 library and sequenced, and the deduced amino acid sequence was studied. The predicted mature protein is a type I membrane protein of 597 amino acids with five potential N-glycosylation sites. There are five disulfide-bonded, extracellular, immunoglobulin superfamily domains (two variable-region set and three constant-region set), a single hydrophobic, membrane-spanning domain, and a cytoplasmic domain of 59 residues. The overall structure is similar to that of the human tumor marker MUC 18 and the chicken neural adhesion molecule SC1. The extracellular domains and cytoplasmic domain contain consensus motifs for the binding of integrin and Src homology 3 domains, respectively, suggesting possible receptor and signal-transduction function. Immunostaining of human tissues demonstrated a wide distribution and provided evidence that the glycoprotein is under developmental control in liver and may also be regulated during differentiation in other tissues.

Basis for selection of improved carbohydrate-binding single-chain antibodies from synthetic gene libraries.

A technique is described for the simultaneous and controlled random mutation of all three heavy or light chain complementarity-determining regions (CDRs) in a single-chain Fv specific for the O polysaccharide of Salmonella serogroup B. Sense oligonucleotides were synthesized such that the central bases encoding a CDR were randomized by equimolar spiking with A, G, C, and T at a level of 10% while the antisense strands contained inosine in the spiked regions. Phage display of libraries assembled from the spiked oligonucleotides by a synthetic ligase chain reaction demonstrated a bias for selection of mutants that formed dimers and higher oligomers. Kinetic analyses showed that oligomerization increased association rates in addition to slowing dissociation rates. In combination with some contribution from reduced steric clashes with residues in heavy-chain CDR2, oligomerization resulted in functional affinities that were much higher than that of the monomeric form of the wild-type single-chain Fv.

Analysis of the mouse Splotch-delayed mutation indicates that the Pax-3 paired domain can influence homeodomain DNA-binding activity.

The murine Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain, and alterations in the Pax-3 gene are responsible for the neural tube defects observed in the Splotch (Sp) mouse mutant. Of five Sp alleles, Splotch-delayed (Spd) is the only one that encodes a full-length Pax-3 protein, containing a single glycine-to-arginine substitution within the paired domain. To better understand the consequence of this mutation on Pax-3 function, we have analyzed the DNA-binding properties of wild-type and Spd Pax-3, using oligonucleotides that bind primarily to the paired domain (e5) or exclusively to the homeodomain (P2). Wild-type Pax-3 was found to bind e5 in a specific manner. In contrast, the Spd mutation reduced binding of Pax-3 to e5 17-fold, revealing a defect in DNA binding by the paired domain. Surprisingly, the Spd mutation also drastically reduced the homeodomain-specific binding to P2 by 21-fold when compared with the wild-type protein. Interestingly, a deletion which removes the Spd mutation was found to restore P2-binding activity, suggesting that within the full-length Pax-3 protein, the paired domain and homeodomain may interact. We conclude, therefore, that the Spd mutation is phenotyically expressed in vitro by a defect in the DNA-binding properties of Pax-3. Furthermore, it is apparent that the paired domain and homeodomain of Pax-3 do not function as independent domains, since a mutation in the former impairs the DNA-binding activity of the latter.

Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol.

Antisense oligodeoxyribonucleotides targeted to the epidermal growth factor (EGF) receptor were encapsulated into liposomes linked to folate via a polyethylene glycol spacer (folate-PEG-liposomes) and efficiently delivered into cultured KB cells via folate receptor-mediated endocytosis. The oligonucleotides were a phosphodiester 15-mer antisense to the EGF receptor (EGFR) gene stop codon (AEGFR2), the same sequence with three phosphorothioate linkages at each terminus (AEGFR2S), a randomized 15-mer control of similar base composition to AEGFR2 (RC15), a 14-mer control derived from a symmetrized Escherichia coli lac operator (LACM), and the 5'-fluorescein-labeled homologs of several of the above. Cellular uptake of AEGFR2 encapsulated in folate-PEG-liposomes was nine times higher than AEGFR2 encapsulated in nontargeted liposomes and 16 times higher than unencapsulated AEGFR2. Treatment of KB cells with AEGFR2 in folate-PEG-liposomes resulted in growth inhibition and significant morphological changes. Curiously, AEGFR2 and AEGFR2S encapsulated in folate-PEG-liposomes exhibited virtually identical growth inhibitory effects, reducing KB cell proliferation by > 90% 48 hr after the cells were treated for 4 hr with 3 microM oligonucleotide. Free AEGFR2 caused almost no growth inhibition, whereas free AEGFR2S was only one-fifth as potent as the folate-PEG-liposome-encapsulated oligonucleotide. Growth inhibition of the oligonucleotide-treated cells was probably due to reduced EGFR expression because indirect immunofluorescence staining of the cells with a monoclonal antibody against the EGFR showed an almost quantitative reduction of the EGFR in cells treated with folate-PEG-liposome-entrapped AEGFR2. These results suggest that antisense oligonucleotide encapsulation in folate-PEG-liposomes promise efficient and tumor-specific delivery and that phosphorothioate oligonucleotides appear to offer no major advantage over native phosphodiester DNA when delivered by this route.

The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.

In vivo gene expression: DNA electrotransfer

The use of electric pulses to deliver therapeutic molecules to tissues and organs in vivo is a rapidly growing field of research. Electrotransfer can be used to deliver a wide range of potentially therapeutic agents, including drugs, proteins, oligonucleotides, RNA and DNA. Optimization of this approach depends upon a number of parameters such as target organ accessibility, cell turnover, microelectrode design, electric pulsing protocols and the physiological response to the therapeutic agent. Many organs have been successfully transfected by electroporation, including skin, liver, skeletal and cardiac muscle, male and female germ cells, artery, gut, kidney, retinal ganglion cells, cornea, spinal cord, joint synovium and brain. Electrotransfer technology is relevant in a variety of research and clinical settings including cancer therapy, modulation of pathogenic immune reactions, delivery of therapeutic proteins and drugs, and the identification of drug targets by the modulation of normal gene expression. This, together with the capacity to deliver very large DNA constructs, greatly expands the research and clinical applications of in vivo DNA electrotransfer.

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at similar to3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 mug/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (chi = 81(.)51%) was significantly better (P = 0(.)0036) than those that possessed 4 positive charges (chi = 56(.)8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P < 0(.)0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases. (C) 2004 Elsevier Ltd. All rights reserved.

Versatile peptide dendrimers for nucleic acid delivery

Dendrimers are nonviral vectors that have attracted interest on account of a number of features. They are structurally versatile because their size, shape, and surface charge can be selectively altered. Here we examine the functions of a new family of composite dendrimers that were synthesized with lipidic amino acid cores. These dendrimers are bifunctional because they are characterized by positively charged (lysine) modules for interaction with nucleic acids and neutral lipidic moieties for membrane lipid-bilayer transit. We assessed their structure-function correlations by a combination of molecular and biophysical techniques. Our assessment revealed an unexpected pleitropy of functions subserved by these vectors that included plasmid and oligonucleotide delivery. We also generated a firefly luciferase cell line in which we could modulate luciferase activity by RNA interference. We found that these vectors could also mediate RNA suppression of luciferase expression by delivering double-stranded luciferase transcripts generated in vitro. The structural uniqueness of these lipidic peptide dendrimers coupled with their ease and specificity of assembly and the versatility in their choice of cargo, puts them in a new category of macromolecule carriers. These vectors, therefore, have potential applications as epigenetic modifiers of gene function. (C) 2004 Wiley-Liss, Inc. and the American Pharmacists Association.

Development of an oligonucleotide-based SNP detection method on lateral flow strips using hexapet tages

Detection of point mutations or single nucleotide polymorphisms (SNPs) is important in relation to disease susceptibility or detection in pathogens of mutations determining drug resistance or host range. There is an emergent need for rapid detection methods amenable to point-of-care applications. The purpose of this study was to reduce to practice a novel method for SNP detection and to demonstrate that this technology can be used downstream of nucleic acid amplification. The authors used a model system to develop an oligonucleotide-based SNP detection system on nitrocellulose lateral flow strips. To optimize the assay they used cloned sequences of the herpes simplex virus-1 (HSV-1) DNA polymerase gene into which they introduced a point mutation. The assay system uses chimeric polymerase chain reaction (PCR) primers that incorporate hexameric repeat tags ("hexapet tags"). The chimeric sequences allow capture of amplified products to predefined positions on a lateral flow strip. These "hexapet" sequences have minimal cross-reactivity and allow specific hybridization-based capture of the PCR products at room temperature onto lateral flow strips that have been striped with complementary hexapet tags. The allele-specific amplification was carried out with both mutant and wild-type primer sets present in the PCR mix ("competitive" format). The resulting PCR products carried a hexapet tag that corresponded with either a wild-type or mutant sequence. The lateral flow strips are dropped into the PCR reaction tube, and mutant sequence and wild-type sequences diffuse along the strip and are captured at the corresponding position on the strip. A red line indicative of a positive reaction is visible after 1 minute. Unlike other systems that require separate reactions and strips for each target sequence, this system allows multiplex PCR reactions and multiplex detection on a single strip or other suitable substrates. Unambiguous visual discrimination of a point mutation under room temperature hybridization conditions was achieved with this model system in 10 minutes after PCR. The authors have developed a capture-based hybridization method for the detection and discrimination of HSV-1 DNA polymerase genes that contain a single nucleotide change. It has been demonstrated that the hexapet oligonucleotides can be adapted for hybridization on the lateral flow strip platform for discrimination of SNPs. This is the first step in demonstrating SNP detection on lateral flow using the hexapet oligonucleotide capture system. It is anticipated that this novel system can be widely used in point-of-care settings.

Zebrafish KLF4 is essential for anterior mesendoderm/pre-polster differentiation and hatching

Gene knockout studies of Kruppel-like factors (KLFs) in mice have shown essential roles in organogenesis. A screen for KLF family members in zebrafish identified many KLFs. One of these, zebrafish KLF4 (zKLF4) is the homologue of neptune, a Xenopus laevis KLF. zKLF4 is expressed from approximately 80% epiboly a patch of dorsal/anterior mesendodermal cells called the pre-polster and, subsequently, in the polster and hatching gland. Here we investigate the function of zKLF4 using morpholino-based antisense oligonucleotides. Knockdown of zKLF4 resulted in complete absence of hatching gland formation and subsequent hatching in zebrafish. In addition, there was early knockdown of expression of the pre-polster/anterior mesendoderm markers CatL, cap1, and BMP4. These results indicate zKLF4 is expressed within the pre-polster, an early mesendodermal site, and that it plays a critical role in the differentiation of these cells into hatching gland cells.

Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development A - Possible role for AHCYL1 in inositol phospholipid signaling

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with similar to 50% protein identity to adenosyl homocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs(zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.

DNA Motifs Suppressing TLR9 Responses

Immune cells respond to bacterial DNA containing unmethylated CpG motifs via Toll-like receptor 9 (TLR9). Given the apparent role of TLR9 in development of systemic lupus erythernatosus (SLE), there is interest in the development of TLR9 inhibitors. TLR9-mediated responses are reported to be inhibited by a confusing variety of different DNA sequences and structures. To aid characterization, we have provisionally categorized TLR9-inhibitory oligodeoxynucleoti des (ODN) into 4 classes, on the basis of sequence and probable mode of action. Class I are short G-rich ODN, which show sequence-specific inhibition of all TLR9 responses, and may be direct competitive inhibitors for DNA binding to TLR9. Class II are telomeric repeat motifs that inhibit STAT signaling, and thus are not specific to TLR9 responses. Because Class II ODN are generally made as 24-base phosphorothioate-modified ODN (PS-ODN), they also fall into Class IV, defined as long PS-ODN, which inhibit TLR9 responses in a sequence-nonspecific manner. Class III includes oligo (dG) that forms a 4-stranded structure and inhibits DNA uptake. The Class I G-rich motifs show the most promise as selective and potent TLR9 inhibitors for therapeutic applications.