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.
A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and ...bind to their complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding deoxyribooligonucleotides and that they cannot function as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers.
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).
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.
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.
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.
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.
The number of optical coherence tomography (OCT) examinations in substance use disorders is gradually increasing. However, OCT findings in opioid use disorder (OUD) have not yet been investigated. In ...this study, we compared the retinal nerve fiber layer (RNFL), the ganglion cell layer (GCL), the inner plexiform layer (IPL), and choroid thickness (CT) of OUD and control groups. We included 43 male patients and 43 healthy male controls of similar age (p = 0.296) in the study, prospectively. On the day of OCT application, urine toxic screening test results of all OUD patients were positive for opioid use. There was a significant difference between OUD and control groups in terms of CT (p < 0.05), nasal superior (NS), and nasal (N) sectors of the RNFL (p < 0.05) values of both eyes. According to the binary logistic regression analysis, the sensitivity of mean NS (p = 0.001) and mean CT (p = 0.007) related to the diagnosis of OUD was 72.1 percent, and the specificity was 65.1 percent. Receiver operating characteristic (ROC) analysis revealed that the sensitivity and specificity of mean CT for the diagnosis of OUD were 18.6% and 97.7%, respectively. This is the first study to investigate the OCT findings in OUD. Our findings are important in terms of showing thinning in the choroidal layer and an increase in the volume of the NS and N sectors of RNFL while detecting opioids in the body/urine. Further studies are needed to clarify whether these differences are due to the acute and/or chronic effects of opioids.
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.
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•RNA duplexes are on average 4 °C higher than for PNA•DNA duplexes and follow quantitatively the same variation with mismatches as do the PNA•DNA duplexes. Also a faster k a and a slower k d are found for PNA•RNA duplexes compared to the PNA•DNA duplexes. An overall fair correlation between T m, k a, and k d is found for a series of PNA•DNA and PNA•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.
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 A
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 μL of whole blood.
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