Hairpin structures formed by alpha satellite DNA of human centromeres are cleaved by human topoisomerase IIalpha.

Although centromere function has been conserved through evolution, apparently no interspecies consensus DNA sequence exists. Instead, centromere DNA may be interconnected through the formation of certain DNA structures creating topological binding sites for centromeric proteins. DNA topoisomerase II is a protein, which is located at centromeres, and enzymatic topoisomerase II activity correlates with centromere activity in human cells. It is therefore possible that topoisomerase II recognizes and interacts with the alpha satellite DNA of human centromeres through an interaction with potential DNA structures formed solely at active centromeres. In the present study, human topoisomerase IIalpha-mediated cleavage at centromeric DNA sequences was examined in vitro. The investigation has revealed that the enzyme recognizes and cleaves a specific hairpin structure formed by alpha satellite DNA. The topoisomerase introduces a single-stranded break at the hairpin loop in a reaction, where DNA ligation is partly uncoupled from the cleavage reaction. A mutational analysis has revealed, which features of the hairpin are required for topoisomerease IIalpha-mediated cleavage. Based on this a model is discussed, where topoisomerase II interacts with two hairpins as a mediator of centromere cohesion.

Assembly and structural analysis of a covalently closed nano-scale DNA cage.

The inherent properties of DNA as a stable polymer with unique affinity for partner molecules determined by the specific Watson-Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnology. Here we present the design, construction and structural analysis of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helices that assembles from eight oligonucleotides with a yield of approximately 30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-molecules to enable their investigation in certain harmful environments or even allow their organization into higher order structures.

Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis.

BACKGROUND: In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature. METHODS: Synchronized cultured cells were fixed with methanol/acetic acid to prepare chromosome spreads in teflon-coated diagnostic well-slides. Apart from the slide format and the chromosome spreading everything was done essentially according to standard protocols. Hybridization targets were detected in situ with padlock probes, which were ligated and amplified using target primed rolling circle DNA synthesis, and detected by fluorescence labeling. RESULTS: An optimized protocol for the spreading of condensed metaphase chromosomes in teflon-coated diagnostic well-slides was developed. Applying this protocol we generated specimens for target primed rolling circle DNA synthesis of padlock probes recognizing a 40 nucleotide sequence in the male specific repetitive satellite I sequence (DYZ1) on the Y-chromosome and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a) gene positioned on the long arm of chromosome 6. These targets were detected with good efficiency, but the efficiency on other target sites was unsatisfactory. CONCLUSION: Our aim was to test the applicability of the method used on mitochondrial DNA to the analysis of nuclear genomes, in particular as represented by metaphase spreads. An optimized protocol for chromosome spreading in diagnostic well-slides was used for the detection of circularized padlock probes amplified by target primed rolling circle DNA synthesis from condensed metaphase chromosomes. We were able to detect a 40 nucleotide sequence in the male specific repetitive satellite I sequence and a 32 nucleotide sequence in the repetitive kringle IV domain in the apolipoprotein(a) gene. Our overall conclusion is that whilst this type of reaction indeed can be brought to work on nuclear genomes, including metaphase chromosomes, the total efficiency of this multistep reaction is at present relatively low (1-10% of target sites picked up), meaning that it is best suited for the detection of targets that exist in multiple copies per cell. Changing this will require substantial efforts to systematically increase the efficiency in each step.

A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis.

BACKGROUND: The quality of chemically synthesized oligonucleotides falls with the length of the oligonucleotide, not least due to depurinations and premature termination during production. This limits the use of long oligonucleotides in assays where long high-quality oligonucleotides are needed (e.g. padlock probes). Another problem with chemically synthesized oligonucleotides is that secondary structures contained within an oligonucleotide reduce the efficiency of HPLC and/or PAGE purification. Additionally, ligation of chemically synthesized oligonucleotides is less efficient than the ligation of enzymatically produced DNA molecules. RESULTS: Chemically synthesized oligonucleotides with hairpin structures were acquired from our standard supplier. The stem of the hairpin contained recognition sequences for the Nt. Alw I nicking enzyme and the Mly I restriction enzyme. These double stranded regions were positioned in a way to allow self-templated circularization of the oligonucleotide. Following ligation, tandem repeats of the complementary sequence of the circular oligonucleotide could be produced through rolling circle DNA synthesis. By running successive rounds of ligation, rolling circle DNA synthesis, and nicking, the original oligonucleotide could be amplified as either the (+)-strand or the (-)-strand. Alternatively, the hairpin structure could be removed by cleavage with the Mly I restriction enzyme, thereby releasing the oligonucleotide sequence contained within the hairpin structure from the hairpin. CONCLUSION: We present here a method for the enzymatic production through DNA amplification of oligonucleotides with freely designable 5'-ends and 3'-ends, using hairpin-containing self-templating oligonucleotides. The hairpin comprises recognition sequences for a nicking enzyme and a restriction enzyme. The oligonucleotides are amplified by successive rounds of ligation, rolling circle DNA synthesis and nicking. Furthermore, the hairpin can be removed by cleavage with the Mly I restriction enzyme. We have named such hairpin structures "suicide cassettes".

In situ detection of non-polyadenylated RNA molecules using Turtle Probes and target primed rolling circle PRINS.

BACKGROUND: In situ detection is traditionally performed with long labeled probes often followed by a signal amplification step to enhance the labeling. Whilst short probes have several advantages over long probes (e.g. higher resolution and specificity) they carry fewer labels per molecule and therefore require higher amplification for detection. Furthermore, short probes relying only on hybridization for specificity can result in non-specific signals appearing anywhere the probe attaches to the target specimen. One way to obtain high amplification whilst minimizing the risk of false positivity is to use small circular probes (e.g. Padlock Probes) in combination with target primed rolling circle DNA synthesis. This has previously been used for DNA detection in situ, but not until now for RNA targets. RESULTS: We present here a proof of principle investigation of a novel rolling circle technology for the detection of non-polyadenylated RNA molecules in situ, including a new probe format (the Turtle Probe) and optimized procedures for its use on formalin fixed paraffin embedded tissue sections and in solid support format applications. CONCLUSION: The method presented combines the high discriminatory power of short oligonucleotide probes with the impressive amplification power and selectivity of the rolling circle reaction, providing excellent signal to noise ratios in combination with exact target localization due to the target primed reaction. Furthermore, the procedure is easily multiplexed, allowing visualization of several different RNAs.

A different approach to telomere analysis with ddPRINS in chronic lymphocytic leukemia.

Telomeric sequences, located at the very end of the chromosomes, compensate for the chromosomal shortening as it happens after each round of cell division. Telomeric sequences influence the progress of cellular senescence and cancer progression. It has been reported that telomeres are shortened in acute leukemias where the cell turnover is high. B-cell chronic lymphocytic leukemia (CLL) is a particularly interesting haematological malignancy in regard to telomere dynamics because most of the malignant cells in CLL are mitotically inactive. In this study, we analysed the telomere length in patients with B-cell CLL in a comparison with the control group by using ddPRINS technique. Twenty patients with CLL and four healthy donors as a control group were included. We found short telomeres and no detectable telomeric repeats at the sites of chromosome fusion. We hypothesise that the telomeric erosion in CLL may reflect the dominance of malignant cells with an abnormally long life span. These cells may have encountered many antigenic stimulants in the past and hence underwent multiple clonal expansions. Our findings imply that shortened telomeres in CLL may be reflecting the "history" of the disease and serve as an independent prognostic factor.

Single-copy and point mutation analysis (rolling-circle PRINS).

This protocol is a prototype for a series of upcoming procedures aimed at the detection of single molecules of nucleic acids in situ. It uses circular probes that, upon hybridization to their target molecule, can template rolling-circle DNA synthesis, if the target also provides a primer for initiation of the DNA synthesis. This primer may be endogenous or artificial, for example, obtained through cleavage with a restriction enzyme. In this format, the primed in situ labeling (PRINS) product takes the form of a long-tandem repeat copy of the probe, covalently attached to the site of synthesis, and tagged it with copies of the probe. The amplification is sufficient for the detection of single, oligonucleotide size, targets and, depending on the probe format, the probe may detect variations in the targets down to single base substitutions.

Detection and sizing of telomeric and other simple repeats by dideoxy-PRINS.

The protocol is suited for the quantitative and qualitative detection of simple repeat target DNA composed of three or fewer of the four bases A, C, G, and T. A consequence of the lacking base(s) is that such DNA can be synthesized from nucleotide mixtures containing the particular bases as dideoxynucleotides. Most genomic DNA contains all four bases and can therefore not be synthesized from such a nucleotide mixture. The combined effects of probe specificity and selective DNA synthesis from the nucleotide mixture improves the signal-to-noise ratio for such targets approximately an order of magnitude, enabling the detection of exceedingly small hybridization targets (e.g., variant telomeric repeat variants embedded in, or situated next to, the main repeat), provided that they present enough template for the DNA synthesis.

Improved diagnosis of mycobacterial infections in formalin-fixed and paraffin-embedded sections with nested polymerase chain reaction.

Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.

Recurrent genomic imbalances in B-cell splenic marginal-zone lymphoma revealed by comparative genomic hybridization.

The cytogenetics of splenic marginal zone lymphoma (SMZL) is less well characterized than the cytogenetics of other non-Hodgkin B-cell lymphomas. The aim of this study was to address this issue by identifying characteristic copy number imbalances in SMZL, for which purpose we analyzed 20 SMZL cases by comparative genomic hybridization (CGH), adding chromosome banding and fluorescence in situ hybridization (FISH) in some cases. CGH identified copy number imbalances in 70% of the cases. Imbalances were recurrently observed for chromosomes 3 (20%), 6 (20%), 7 (25%), 12 (20%), and 14 (10%). The minimally involved regions of these chromosomes were gains of 3q25 approximately qter and 12q13 approximately q15, and loss of 6q23, 7q31, and 14q22 approximately q24. A compilation of our data with data from 3 previous SMZL CGH studies revealed a significant heterogeneity between the studies. Eleven imbalances were recurrently observed in the compiled data set, as opposed to only 5 in our data set. The most frequently observed imbalances in the 73 SMZL cases of the compiled data set were gains of 3q (27%) and 12q (15%), and loss of 7q (18%). Our data suggest that SMZL constitute a genetically heterogeneous disease where gain of 3q25 and loss of 7q31 are the most likely imbalances to be involved in the pathogenesis of the disease.

Telomerase activity in human leukemic cells with or without monosomy 7 or 7q-.

BACKGROUND: In bone marrow material from patients with various leukemias we noted that samples with either a deletion on the long arm of one chromosome 7 (7q-) or a monosomy 7 had a higher telomerase activity. Considering that introduction of a chromosome 7 into a cancer cell line had been reported to eliminate telomerase activity, that 7q- is a common negative prognostic finding in cancers, and that the deleted segment (band 7q31) contains an unidentified tumor suppressor gene, we wondered if this gene might be a telomerase inhibitor. RESULTS: We found no significant difference in telomerase activity between the three groups of patient samples. In contrast to reports on tumor cell lines we observed no amplification of the telomerase genes. METHODS: We analyzed telomerase activity and copy number of the telomerase genes hTERT and hTR in frozen archival bone marrow samples from leukemia patients with a referral diagnosis of AML, and either a monosomy for chromosome 7, a deletion on the long arm of chromosome 7 (7q-), or none of these aberrations. Telomerase activity was measured with a commercially available kit, and the copy number of the telomerase genes was tested by FISH. CONCLUSIONS: We found no evidence of a telomerase inhibitor in band 7q31. The lack of telomerase gene amplification found in cell lines from solid tumors could reflect that this amplification is a property of solid tumors, not of hematological cancers.

Multiple telomeric aberrations in a telomerase-positive leukemia patient.

Bone marrow samples from a pancytopenia/leukemia patient were routinely analyzed at first and second admission. At the first presentation, the karyotype was normal, whereas 17 months later several chromosome aberrations were recognized including presumed additions to the short arms of chromosomes 1 and 16 in all cells, and numerous other aberrations in subpopulations of cells. From the predominance of aberrations at chromosome ends, we suspected insufficient telomere maintenance as an underlying mechanism behind the karyotype changes, in particular as an interstitial deletion in the region harboring the gene for the RNA component (hTERC) of the telomerase enzyme was also noticed; however, while molecular cytogenetic investigation confirmed the terminal aberrations, we found the malignant cells positive for telomerase activity and the presence of an hTERC gene on both chromosomes 3. A presumed chromosome 1 addition turned out to reflect an amplification of a tandemly repeated sequence element. Labeling of multiple tandem repeat sequences in situ by a novel multicolor primed in situ hybridization showed no evidence of instability of other repeated DNA elements.

Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor.

Real-time detection of enzyme activities may present the easiest and most reliable way of obtaining quantitative analyses in biological samples. We present a new DNA-biosensor capable of detecting the activity of the potential anticancer drug target tyrosyl-DNA phosphodiesterase 1 (TDP1) in a very simple, high throughput, and real-time format. The biosensor is specific for Tdp1 even in complex biological samples, such as human cell extracts, and may consequently find future use in fundamental studies as well as a cancer predictive tool allowing fast analyses of diagnostic cell samples such as biopsies. TDP1 removes covalent 3'DNA adducts in DNA single-strand break repair. This enzymatic activity forms the basis of the design of the TDP1-biosensor, which consists of a short hairpin-forming oligonucleotide having a 5'fluorophore and a 3'quencher brought in close proximity by the secondary structure of the biosensor. The specific action of TDP1 removes the quencher, thereby enabling optical detection of the fluorophore. Since the enzymatic action of TDP1 is the only "signal amplification" the increase in fluorescence may easily be followed in real-time and allows quantitative analyses of TDP1 activity in pure enzyme fractions as well as in crude cell extracts. In the present study we demonstrate the specificity of the biosensor, its ability to quantitatively detect up- or down-regulated TDP1 activity, and that it may be used for measuring and for analyzing the mechanism of TDP1 inhibition.

Droplet microfluidics platform for highly sensitive and quantitative detection of malaria-causing Plasmodium parasites based on enzyme activity measurement.

We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/µL. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings, may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water or food quality control, or other purposes within applied or basic science.

Detection of single enzymatic events in rare or single cells using microfluidics.

In the present study we demonstrate highly sensitive detection of rare, aberrant cells in a population of wild-type human cells by combining a rolling-circle-enhanced enzyme activity single-molecule detection assay with a custom-designed microfluidic device. Besides reliable detection of low concentrations of aberrant cells, the integrated system allowed multiplexed detection of individual enzymatic events at the single cell level. The single cell sensitivity of the presented setup relies on the combination of single-molecule rolling-circle-enhanced enzyme activity detection with the fast reaction kinetics provided by a picoliter droplet reaction volume and subsequent concentration of signals in a customized drop-trap device. This setup allows the fast reliable analyses of enzyme activities in a vast number of single cells, thereby offering a valuable tool for basic research as well as theranostics.

Microfluidics-mediated isothermal detection of enzyme activity at the single molecule level.

Conventional analysis of enzymatic activity, often carried out on pools of cells, is blind to heterogeneity in the population. Here, we combine microfluidics with a previously developed isothermal rolling circle amplification-based assay to investigate multiple enzymatic activities in down to single cells. This microfluidics-meditated assay performs at very high sensitivity in picoliter incubators with small quantities of biological materials. Furthermore, we demonstrate the assay's capability of multiplexed detection of at least three enzyme activities at the single molecule level.

Rapid, sensitive, type specific PCR detection of the E7 region of human papillomavirus type 16 and 18 from paraffin embedded sections of cervical carcinoma.

Human papillomavirus (HPV) infection, and in particularly infection with HPVs 16 and 18, is a central carcinogenic factor in the uterine cervix. We established and optimized a PCR assay for the detection and discrimination of HPV types 16 and 18 in archival formaldehyde fixed and paraffin embedded (FFPE) sections of cervical cancer.Tissue blocks from 35 cases of in situ or invasive cervical squamous cell carcinoma and surrogate FFPE sections containing the cell lines HeLa and SiHa were tested for HPV 16 and HPV18 by conventional PCR using type specific primers, and for the housekeeping gene beta-actin. Using HPV 16 E7 primers, PCR products with the expected length were detected in 18 of 35 of FFPE sections (51%). HPV 18 E7 specific sequences were detected in 3 of 35 FFPE sections (9%).In our experience, the PCR technique is a robust, simple and sensitive way of type specific detection of HPV16 and HPV18 genes in FFPE tissue. That makes this technique applicable to routine practices of HPV detection.

Structure of nanoscale truncated octahedral DNA cages: variation of single-stranded linker regions and influence on assembly yields.

The assembly, structure, and stability of DNA nanocages with the shape of truncated octahedra have been studied. The cages are composed of 12 double-stranded B-DNA helices interrupted by single-stranded linkers of thymidines of varying length that constitute the truncated corners of the structure. The structures assemble with a high efficiency in a one-step procedure, compared to previously published structures of similar complexity. The structures of the cages were determined by small-angle X-ray scattering. With increasing linker length, there is a systematic increase of the cage size and decrease of the twist angle of the double helices with respect to the symmetry planes of the cage structure. In the present study, we demonstrate the length of the single-stranded linker regions, which impose a certain degree of flexibility to the structure, to be the important determinant for efficient assembly. The linker length can be decreased to three thymidines without affecting assembly yield or the overall structural characteristics of the DNA cages. A linker length of two thymidines represents a sharp cutoff abolishing cage assembly. This is supported by energy minimization calculations suggesting substantial hydrogen bond deformation in a cage with linkers of two thymidines.

p16 as a diagnostic marker of cervical neoplasia: a tissue microarray study of 796 archival specimens.

BACKGROUND: To evaluate the usefulness of this biomarker in the diagnosis of cases of cervical neoplasia we studied the immunohistochemical expression of p16INK4a in a large series of archival cervical biopsies arranged into tissue microarray format. METHODS: TMAs were constructed with tissue cores from archival formalin fixed, paraffin-embedded donor tissues from 796 patients, and included cases of cervical intraepithelial neoplasia (CIN)1 (n = 249), CIN2 (n = 233), CIN3 (n = 181), and invasive cervical carcinoma (n = 133). p16INK4a expression was scored using two different protocols: 1) positive vs negative p16INK4a staining; 2) a semi-quantitative immunohistochemical score (0 to 8 points) according to the intensity of staining and the proportion of stained cells RESULTS: p16INK4A expression was not seen in normal cervix tissue, but was found with increasing frequency in the sequence: CIN1 (180/249; 72.3%) - CIN2 (212/233; 91.0%) - CIN3 (178/181; 98.3%) - invasive carcinoma (131/133; 98.5%). Using semi-quantitative scoring, all normal cervical samples had low scores (from 0 to 2 points), whilst the number of specimens with high scores was proportional to the degree of cervical dysplasia or the presence of invasive carcinoma. CONCLUSION: Immunohistochemical analysis of p16INK4a expression is a useful diagnostic tool. Expression is related to the degree of histological dysplasia, suggesting that it may have prognostic and predicative value in the management of cervical neoplasia.