Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions.

We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T. thermophila snRNAs all have unique 5' ends, which start with an adenine residue. In contrast, with the exception of U6, their 3' ends show some size heterogeneity. The primary sequences of the T. thermophila snRNAs contain the sequence motifs shown, or proposed, to be of functional importance in other organisms. Furthermore, secondary structures closely similar to phylogenetically proven models can be inferred from the T. thermophila data. Analysis of the snRNA sequences identifies three potential snRNA-snRNA base-pairing interactions, all of which are consistent with available phylogenetic data. Two of these occur between U2 and U6, whereas the third occurs between U1 and U2. The proposed interactions locate the intron 5' splice-site close to the intron branch-site nucleotide as well as to the most highly conserved domain of U6. We envisage that these interactions may facilitate the first step of pre-mRNA splicing.

Structural organization of the genes encoding the small nuclear RNAs U1 to U6 of Tetrahymena thermophila is very similar to that of plant small nuclear RNA genes.

We report the sequences of the genes encoding the small nuclear RNAs (snRNAs) U1 to U6 of the ciliate Tetrahymena thermophila. The genes of the individual snRNAs exist in two to six slightly different copies per haploid genome. Sequence analyses of the gene-flanking regions indicate that there are two classes of snRNA genes. Both classes are characterized by several conserved sequence elements, some of which are unique to each class and some of which are found in both classes. Comparison of the promoter structure of the snRNA genes of T. thermophila with the promoter structures of snRNA genes of other organisms revealed several similarities to plant snRNA genes. These similarities include the overall promoter architecture as well as specific sequence elements. The structural organization of the 3' flanking region of some of the T. thermophila snRNA genes is not observed in other organisms. This finding is discussed in relation to a possible role in snRNA 3'-end formation.

A novel class of nucleolar RNAs from Tetrahymena.

We describe a family of at least four nucleolar RNAs (snoRNAs) from the ciliate, Tetrahymena. The snoRNAs are 120-140 nucleotides long, moderately AU-rich and contain no modified nucleotides. Their 5' ends are blocked by a cap of unknown nature. The snoRNAs can be folded into similar secondary structures consisting of two hairpins separated by a single-stranded AU-rich spacer. The sequences and secondary structures show no extensive sequence or secondary structure resemblance to any other small RNAs in the public databases.

PNA-mediated purification of PCR amplifiable human genomic DNA from whole blood.

A simple and rapid procedure whereby human genomic DNA can be purified in a PCR amplifiable form from whole blood is described. In a first step, human genomic DNA is hybridized in solution to a biotinylated peptide nucleic acid (PNA), which forms a high-affinity triplex with A7 sequence motifs in the target DNA. The complex is then captured onto paramagnetic streptavidin-coated particles, which are subsequently transferred directly into the PCR. The purification method effectively removes inhibitors of the PCR from as much as 500 microL of whole blood.

Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection.

Using an oligohistidine peptide nucleic acids (oligohistidine-PNA) chimera, we have developed a rapid hybrid selection method that allows efficient, sequence-specific purification of a target nucleic acid. The method exploits two fundamental features of PNA. First, that PNA binds with high affinity and specificity to its complementary nucleic acid. Second, that amino acids are easily attached to the PNA oligomer during synthesis. We show that a (His)6-PNA chimera exhibits strong binding to chelated Ni2+ ions without compromising its native PNA hybridization properties. We further show that these characteristics allow the (His)6-PNA/DNA complex to be purified by the well-established method of metal ion affinity chromatography using a Ni(2+)-NTA (nitrilotriactic acid) resin. Specificity and efficiency are the touchstones of any nucleic acid purification scheme. We show that the specificity of the (His)6-PNA selection approach is such that oligonucleotides differing by only a single nucleotide can be selectively purified. We also show that large RNAs (2224 nucleotides) can be captured with high efficiency by using multiple (His)6-PNA probes. PNA can hybridize to nucleic acids in low-salt concentrations that destabilize native nucleic acid structures. We demonstrate that this property of PNA can be utilized to purify an oligonucleotide in which the target sequence forms part of an intramolecular stem/loop structure.

Locked nucleic acids: a promising molecular family for gene-function analysis and antisense drug development.

Locked nucleic acids (LNAs) are a family of conformationally locked oligonucleotide analogs inducing unprecedented binding affinity towards DNA/RNA target sequences. Importantly, by virtue of the structural resemblance of LNAs to natural nucleic acid monomers, a combination of LNA chemistry with other oligonucleotide chemistries can be exploited to fine-tune the properties towards optimized antisense drug development and target validation technology. The first promising antisense results from experiments with LNA in living animals are described.

The novel antisense Bcl-2 inhibitor SPC2996 causes rapid leukemic cell clearance and immune activation in chronic lymphocytic leukemia.

SPC2996 is a novel locked nucleic acid phosphorothioate antisense molecule targeting the mRNA of the Bcl-2 oncoprotein. We investigated the mechanism of action of SPC2996 and the basis for its clinically observed immunostimulatory effects in chronic lymphocytic leukemia (CLL). Patients with relapsed CLL were treated with a maximum of six doses of SPC2996 (0.2-6 mg/kg) in a multicenter phase I trial. Microarray-based transcriptional profiling of circulating CLL cells was carried out before and after the first infusion of SPC2996 in 18 patients. Statistically significant transcriptomic changes were observed at doses 4 mg/kg and occurred as early as 24 h after the first infusion of the oligonucleotide. SPC2996 induced the upregulation of 466 genes including a large number of immune response and apoptotic regulator molecules, which were enriched for Toll-like receptor response genes. Serum measurements confirmed the release of pro-inflammatory cytokines including chemokine (C-C motif) ligand 3 (macrophage inflammatory protein 1α) and tumor necrosis factor-α, thereby validating the in vivo transcriptomic data at the protein level. SPC2996 caused a 50% reduction of circulating lymphocytes in five of 18 (28%) patients, which was found to be independent of its immunostimulatory and anti-Bcl-2 effects.

Secondary amyloidosis causing nephrotic syndrome in a patient with non-Hodgkin's lymphoma: quite a rare diagnosis.

Secondary amyloidosis is usually a complication of chronic inflammation. Amyloidosis cases during the course of non-Hodgkin's lymphoma (NHL) are usually of AL-type, only one NHL patient with secondary amyloidosis has been reported. Our 79-year-old male patient visited us with multiple lymphadenopathies, and he was diagnosed with nodal marginal zone B-cell lymphoma. After four cycles of combined chemotherapy; his urea, creatinine levels started to increase and he developed nephrotic-range proteinuria. His rectal biopsy demonstrated amyloid deposition in submucosal vessel walls. The patient has been under hemodialysis for 10 months and his lymphoma is still in partial remission. We presented this case because it is the second NHL patient who developed secondary amyloidosis during his disease course.

Purification of nucleic acids by hybridization to affinity tagged PNA probes.

The use of affinity tagged PNA capture probes offers an efficient means for the purification of nucleic acids by hybridization. Two different approaches are described. A sequence specific method and a generic method. The sequence specific method requires sequence information on the target and synthesis of a dedicated PNA. It can be used to selectively purify the nucleic acid containing the target from non-related nucleic acids and other cellular components. The generic method uses a "universal" triplex forming PNA and requires no sequence information on the target. It can be used in the bulk purification of large nucleic acids.

PCR clamping.

An efficient, PCR based method for the selective amplification of DNA target sequences that differs by a single base pair is described. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.

Expression in E. coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides.

The active sites of the enzyme phenylalanine ammonia-lyase (Pal) from Rhodosporidium toruloides contains a dehydroalanine residue that is believed to be essential for catalytic activity. Furthermore, the dehydroalanine is believed to be added post-translationally as part of a prosthetic group covalently attached to the enzyme. Perhaps for this reason no attempts to produce Pal in foreign host cells have been reported. We have inserted the entire uninterupted pal gene from R. toruloides into the Escherichia coli expression vector pKK 223-3. E. coli cells containing this vector synthesize a protein of the expected size, and extracts prepared from these cells contain a Pal-like activity. The potential implications of this finding are discussed.

A novel, single plasmid, approach to in-frame fusion.

The sequence GCGCGCGCGC contains three overlapping, mutually exclusive, BssHII restriction sites, each corresponding to one of the three different reading frames. When inserted into a plasmid and digested with BssHII, the three sites in this sequence are cleaved at an approximate ratio of 2:1:2. Consequently, this system can be used to simplify in vitro in frame fusions to involve a single plasmid. We have constructed such a plasmid and used it to select an open reading frame in a 1.6 kb cDNA fragment.

PNA synthesis using a novel Boc/acyl protecting group strategy.

The synthesis of novel Boc/acyl protected monomers for the synthesis of peptide nucleic acid (PNA) is described. The oligomerization protocol using these new monomers has been optimized with regard to coupling reagents. The use of base-labile acyl protecting groups at the exocyclic amines of the heterocyclic bases (isobutyryl for guanine and benzoyl for adenine and cytosine) and a PAM-linked solid support offers an attractive alternative to the present procedures used in PNA synthesis. This strategy has been applied for the synthesis of a test 17mer PNA on both control pore glass (CPG) and a polystyrene MBHA support and was used in the preparation of PNA-DNA chimeras.

Detection of thymine [2+2] photodimer repair in DNA: selective reaction of KMnO4.

The specific reaction of potassium permanganate with thymine in single-stranded DNA was employed to analyze thymine [2+2] dimer repair in DNA and in DNA/peptide nucleic acid hybrid duplexes. This simple and highly sensitive chemical assay is convenient for monitoring repair of thymine dimers in oligonucleotides.

Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique.

The binding of a mixed-sequence pentadecamer PNA (peptide nucleic acid) containing all four nucleobases to the fully complementary as well as various singly mismatched RNA and DNA oligonucleotides has been systematically investigated using thermal denaturation and BIAcore surface-interaction techniques. The rate constants for association (k(a)) and dissociation (k(d)) of the duplex formation as well as the thermal stability (melting temperature, T(m)) of the duplexes have been determined. Upon binding to PNA tethered via a biotin-linker to streptavidin at the dextran/gold surface, DNA and RNA sequences containing single mismatches at various positions in the center resulted in increased dissociation and decreased association rate constants. T(m) values for PNA x RNA duplexes are on average 4 degrees C higher than for PNA x DNA duplexes and follow quantitatively the same variation with mismatches as do the PNA x DNA duplexes. Also a faster k(a) and a slower k(d) are found for PNA x RNA duplexes compared to the PNA x DNA duplexes. An overall fair correlation between T(m), k(a), and k(d) is found for a series of PNA x DNA and PNA x RNA duplexes although the determination of k(a) seemed to be prone to artifacts of the method and was not considered capable of providing absolute values representing the association rate constant in bulk solution.

Peptide nucleic acid (PNA)/DNA hybrid duplexes: intercalation by an internally linked anthraquinone.

Peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson-Crick base pairing. A set of reporter compounds that bind to DNA by intercalation are known, but these compounds do not intercalate in PNA/DNA hybrid duplexes. Analysis of the hybrid PNA duplexes requires development of reporter compounds that probe their chemical and physical properties. We prepared a series of anthraquinone (AQ) derivatives that are linked to internal positions of a PNA oligomer. These are the first non-nucleobase functional groups that have been incorporated into a PNA. The resulting PNA(AQ) conjugates form stable hybrids with complementary DNA oligomers. We find that when the AQ groups are covalently bound to PNA that they stabilize the hybrid duplex and are, at least partially, intercalated.

Detection of the factor V Leiden mutation by direct allele-specific hybridization of PCR amplicons to photoimmobilized locked nucleic acids.

BACKGROUND: Individuals carrying the factor V Leiden mutation have been shown to have an increased risk of developing venous thromboembolism. Our aim was to develop an ELISA-like assay to detect the mutation in PCR-amplified genomic DNA using novel, high-affinity DNA analogs, termed locked nucleic acids (LNAs). METHODS: LNA octamer probes complementary to the factor V wild-type or mutated sequence were covalently attached to individual wells of a microtiter plate. Biotinylated factor V amplicons were added, and hybridization to the immobilized LNA probes was scored colorimetrically using a horseradish peroxidase-anti-biotin Fab conjugate and tetramethylbenzidine substrate. RESULTS: In a prospective study of 53 patients, the assay reproducibly scored both factor V homozygotes and heterozygotes with excellent sensitivity and specificity. All results were in complete agreement with the results obtained with the conventional PCR-restriction fragment length polymorphism technique. CONCLUSIONS: The simplicity of the assay and its procedural relatedness to the widely used ELISA format should make it useful for routine factor V testing in the clinical laboratory.

Peptide nucleic acid-anthraquinone conjugates: strand invasion and photoinduced cleavage of duplex DNA.

A bis-peptide nucleic acid (PNA)-anthraquinone imide (AQI) conjugate has been synthesized and shown to form strand invasion complexes with a duplex DNA target. The two arms of the bis-PNA each consist of five consecutive thymine residues and are linked by a flexible, hydrophilic spacer. Probing with potassium permanganate reveals that the bis-PNA complexes to duplex DNA at A5.T5sites with local displacement of the T5DNA strand. The 5 bp sequence targeted by the PNA is the shortest strand invasion complex reported to date. Irradiation of the strand invasion complex results in asymmetric cleavage of the displaced strand, with more efficient cleavage at the 3'-end of the loop. This result indicates that the bis-PNA binds to the DNA such that the C-terminal T5sequence forms the strand invasion complex, leaving the N-terminal T5sequence to bind by triplex formation, thereby placing the AQI closer to the 3'-end of the displaced strand, consistent with the observed photocleavage pattern. The ability of the PNA to directly report its binding site by photoinduced cleavage could have significant utility in mapping the secondary and tertiary structure of nucleic acids.