Kinetics of RNA and RNA:DNA Hybrid Strand Displacement.

In nucleic acid nanotechnology, strand displacement is a widely used mechanism where one strand from a hybridized duplex is exchanged with an invading strand that binds to a toehold, a single-stranded region on the duplex. It is used to perform logic operations on a molecular level, initiate cascaded reactions, or even for in vivo diagnostics and treatments. While systematic experimental studies have been carried out to probe the kinetics of strand displacement in DNA with different toehold lengths, sequences, and mismatch positions, there has not been a comparable investigation of RNA or RNA-DNA hybrid systems. Here, we experimentally study how toehold length, toehold location (5' or 3' end of the strand), and mismatches influence the strand displacement kinetics. We observe reaction acceleration with increasing toehold length and placement of the toehold at the 5' end of the substrate. We find that mismatches closer to the interface of toehold and duplex slow down the reaction more than remote mismatches. A comparison of RNA and DNA displacement with hybrid displacement (RNA invading DNA or DNA invading RNA) is partly explainable by the thermodynamic stabilities of the respective toehold regions, but also suggests that the rearrangement from B-form to A-form helix in the case of RNA invading DNA might play a role in the kinetics.

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

DNA-Specific Biosensors Based on Intramolecular ß-Lactamase-Inhibitor Complex Formation.

Synthetic protein switches that sequence-specifically respond to oligonucleotide-based input triggers provide valuable tools for the readout of oligonucleotide-based biomolecular systems and networks. Here, we discuss a highly modular approach to reversibly control the DNA-directed assembly and disassembly of a complex between TEM1-ß-lactamase and its inhibitor protein BLIP. By conjugating each protein to a unique handle oligonucleotide, the enzyme-inhibitor pair is noncovalently assembled upon the addition of a complementary ssDNA template strand, resulting in inhibition of enzyme activity. Hybridization of an input-oligonucleotide that is complementary to a target recognition sequence in the ssDNA template strand results in the formation of a rigid dsDNA helix that mechanically disrupts the enzyme-inhibitor complex, hereby restoring enzyme activity. Following this noncovalent approach allowed straightforward tuning of the ssDNA template recognition sequence and target oligonucleotide lengths with only a single set of oligonucleotide-functionalized enzyme and inhibitor domains. Using a fluorescent substrate, as little as 10 pM target oligonucleotide resulted in a distinguishable increase in enzyme activity.

Effects of Concentration and Temperature on DNA Hybridization by Two Closely Related Sequences via Large-Scale Coarse-Grained Simulations.

A newly developed coarse-grained model called BioModi is utilized to elucidate the effects of temperature and concentration on DNA hybridization in self-assembly. Large-scale simulations demonstrate that complementary strands of either the tetrablock sequence or randomized sequence with equivalent number of cytosine or guanine nucleotides can form completely hybridized double helices. Even though the end states are the same for the two sequences, there exist multiple kinetic pathways that are populated with a wider range of transient aggregates of different sizes in the system of random sequences compared to that of the tetrablock sequence. The ability of these aggregates to undergo the strand displacement mechanism to form only double helices depends upon the temperature and DNA concentration. On one hand, low temperatures and high concentrations drive the formation and enhance stability of large aggregating species. On the other hand, high temperatures destabilize base-pair interactions and large aggregates. There exists an optimal range of moderate temperatures and low concentrations that allow minimization of large aggregate formation and maximization of fully hybridized dimers. Such investigation on structural dynamics of aggregating species by two closely related sequences during the self-assembly process demonstrates the importance of sequence design in avoiding the formation of metastable species. Finally, from kinetic modeling of self-assembly dynamics, the activation energy for the formation of double helices was found to be in agreement with experimental results. The framework developed in this work can be applied to the future design of DNA nanostructures in both fields of structural DNA nanotechnology and dynamic DNA nanotechnology wherein equilibrium end states and nonequilibrium dynamics are equally important requiring investigation in cooperation.

Homologous PNA Hybridization to Noncanonical DNA G-Quadruplexes.

Potential guanine (G) quadruplex-forming sequences (QFSs) found throughout the genomes and transcriptomes of organisms have emerged as biologically relevant structures. These G-quadruplexes represent novel opportunities for gene regulation at the DNA and RNA levels. Recently, the definition of functional QFSs has been expanding to include a variety of unconventional motifs, including relatively long loop sequences (i.e., >7 nucleotides) separating adjacent G-tracts. We have identified a QFS within the 25S rDNA gene from Saccharomyces cerevisae that features a long loop separating the two 3'-most G-tracts. An oligonucleotide based on this sequence, QFS3, folds into a stable G-quadruplex in vitro. We have studied the interaction between QFS3 and several loop mutants with a small, homologous (G-rich) peptide nucleic acid (PNA) oligomer that is designed to form a DNA/PNA heteroquadruplex. The PNA successfully invades the DNA quadruplex target to form a stable heteroquadruplex, but with surprisingly high PNA:DNA ratios based on surface plasmon resonance and mass spectrometric results. A model for high stoichiometry PNA-DNA heteroquadruplexes is proposed, and the implications for quadruplex targeting by G-rich PNA are discussed.

Kinetics and dynamics of DNA hybridization.

DNA hybridization, wherein strands of DNA form duplex or larger hybrids through noncovalent, sequence-specific interactions, is one of the most fundamental processes in biology. Developing a better understanding of the kinetic and dynamic properties of DNA hybridization will thus help in the elucidation of molecular mechanisms involved in numerous biochemical processes. Moreover, because DNA hybridization has been widely adapted in biotechnology, its study is invaluable to the development of a range of commercially important processes. In this Account, we examine recent studies of the kinetics and dynamics of DNA hybridization, including (i) intramolecular collision of random coil, single-stranded DNA (ssDNA), (ii) nucleic acid hairpin folding, and (iii) considerations of DNA hybridization from both a global view and a detailed base-by-base view. We also examine the spontaneous single-base-pair flipping in duplex DNA because of its importance to both DNA hybridization and repair. Intramolecular collision of random coil ssDNA, with chemical relaxation times ranging from hundreds of nanoseconds to a few microseconds, is investigated both theoretically and experimentally. The first passage time theory of Szabo, Schulten, and Schulten, which determines the average reaction time of the intrachain collision, was tested. Although it was found to provide an acceptable approximation, a more sophisticated theoretical treatment is desirable. Nucleic acid hairpin folding has been extensively investigated as an important model system of DNA hybridization. The relaxation time of hairpin folding and unfolding strongly depends on the stem length, and it may range from hundreds of microseconds to hundreds of milliseconds. The traditional two-state model has been revised to a multistate model as a result of new experimental observations and theoretical study, and partially folded intermediate states have been introduced to the folding energy landscape. On the other hand, new techniques are needed to provide more accurate and detailed information on the dynamics of DNA hairpin folding in the time domain of sub-milliseconds to tens of milliseconds. From a global view, the hybridization of unstructured ssDNA goes through an entropy-controlled nucleation step, whereas the hybridization of ssDNA with a hairpin structure must overcome an extra, enthalpy-controlled energy barrier to eliminate the hairpin. From a detailed base-by-base view, however, there exist many intermediate states. The average single-base-pair hybridization and dehybridization rates in a duplex DNA formation have been determined to be on the order of a millisecond. Meanwhile, accurate information on the early stages of hybridization, such as the dynamics of nucleation, is still lacking. The investigation of spontaneous flipping of a single base in a mismatched base pair in a duplex DNA, although very important, has only recently been initiated because of the earlier lack of suitable probing tools. In sum, the study of DNA hybridization offers a rich range of research opportunities; recent progress is highlighting areas that are ripe for more detailed investigation.

Triplex-forming MicroRNAs form stable complexes with HIV-1 provirus and inhibit its replication.

BACKGROUND: One of the most fascinating discoveries in biology in recent years is unquestionably the identification of the family of small, noncoding RNAs known as microRNAs (miRNAs). Each miRNA targets multiple mRNA species through recognition of complementary sequences, typically located at multiple sites within the 3 untranslated region. In animals, single-stranded miRNA binds specific messenger RNA (mRNA) by a mechanism that is yet to be fully characterized. The bound mRNA remains untranslated resulting in reduced levels of the corresponding protein; however, if the sequence match between the miRNA and its target is precise, the bound mRNA can be degraded resulting in reduced levels of the corresponding transcript. Eukaryotic genes are also regulated by triplex formation between double helix and a third small RNA or DNA molecule. Thousands of triplex-forming (TF) islands in human genomes are mapped. However, the role of TF miRNAs within the hairpin structures of miRNA and the target mRNA has not been reported. We have explored TF complexes between human miRNAs (hsa-miR) that are complementary to human immunodeficiency virus (HIV)-1 and their antiviral potential as therapeutic agents. METHODS: We downloaded mature miRNA sequences from the human miRBase Sequence Database (http://microrna.sanger.ac.uk/sequences/), and computationally analyzed miRNAs that have significant homologies to HIV-1 genome (pNL 4-3 Accession #AF324493). We developed an algorithm to look for triplex-binding motifs (C+CG and T AT) and selected 4 miRNAs with 3 negative controls. TF stability was tested by using fluorophore-labeled duplexes connected by a single hexaethylene glycol moiety, representing HIV-1 proviral motifs, and black-hole quencher-1 labeled oligonucleotides, representing miRNA. RESULTS: Fifty miRNAs were discovered that showed greater than 80% homology to HIV-1, of which 4 hsa-miR that exhibited an ability to form stable triplex with double stranded-HIV-1 sequences were selected. Three negative controls were used. The TF stability of the 4 hsa-miRs and the negative controls were confirmed and measured. Stably transfected Hela-CD4+ cell lines expressing each of the hsa-miR were developed. All 4 miRNAs exhibited a significant inhibition of HIV-1 as measured by HIV-1 p24 enzyme-linked immunosorbent assay (>90%; P>0.001) when compared with the 3 negative controls. By using immunohistochemical staining with triplex binding monoclonal antibodies, significant expression of TF miRNAs was detected in the cell lines, but not in the negative controls (P<0.001). CONCLUSIONS: In this study, we demonstrated for the first time that besides the well-established post-transcriptional silencing based on mRNA degradation, miRNAs may be responsible for long-term latency of HIV-1 by TF, a different mechanism. We provide a possible molecular mechanism by which HIV-1 homologous miRNAs may impart resistance to HIV-1 and suggest a new miRNA-based therapeutic strategy for HIV-1.

Self containment, a property of modular RNA structures, distinguishes microRNAs.

RNA molecules will tend to adopt a folded conformation through the pairing of bases on a single strand; the resulting so-called secondary structure is critical to the function of many types of RNA. The secondary structure of a particular substring of functional RNA may depend on its surrounding sequence. Yet, some RNAs such as microRNAs retain their specific structures during biogenesis, which involves extraction of the substructure from a larger structural context, while other functional RNAs may be composed of a fusion of independent substructures. Such observations raise the question of whether particular functional RNA substructures may be selected for invariance of secondary structure to their surrounding nucleotide context. We define the property of self containment to be the tendency for an RNA sequence to robustly adopt the same optimal secondary structure regardless of whether it exists in isolation or is a substring of a longer sequence of arbitrary nucleotide content. We measured degree of self containment using a scoring method we call the self-containment index and found that miRNA stem loops exhibit high self containment, consistent with the requirement for structural invariance imposed by the miRNA biogenesis pathway, while most other structured RNAs do not. Further analysis revealed a trend toward higher self containment among clustered and conserved miRNAs, suggesting that high self containment may be a characteristic of novel miRNAs acquiring new genomic contexts. We found that miRNAs display significantly enhanced self containment compared to other functional RNAs, but we also found a trend toward natural selection for self containment in most functional RNA classes. We suggest that self containment arises out of selection for robustness against perturbations, invariance during biogenesis, and modular composition of structural function. Analysis of self containment will be important for both annotation and design of functional RNAs. A Python implementation and Web interface to calculate the self-containment index are available at http://kim.bio.upenn.edu/software/.

[Construction of linkage localization lines containing genes with maternal effects].

Since the phenotypes of hybrid progenies involving genes with maternal effects are affected by the genotype of female parent, they cannot reflect the genotypes of individuals. This makes it difficult to develop test cross parents (triple or double recessive lines) for linkage localization and, consequently, hinders the progress in localization researches for these types of genes. In this study, we designed a set of hybridization schemes, the key of which was to make the "maternal-effect" genes homozygous at first, and then the non-maternal-effect genes. Using this scheme, we successfully produced a triple recessive line for genes ch (chocolate), nlw (non-lepis wing) and b-t (maternal brown egg of Tsujitan) on the 13th linkage group and a double recessive line for genes nb (narrow breast) and ki-2 (kidney-shaped egg 2) on the 19th linkage group of Bombyx mori.

Use of a 44k oligoarray to explore the transcriptome of Schistosoma mansoni adult worms.

Recent advances in the study of Schistosoma mansoni genome and transcriptome have led to a better description of the S. mansoni gene complement. In this work, we report the design and use of a new S. mansoni 60-mer oligonucleotide microarray platform with approximately 44,000 probes, based on all publicly available cDNA sequence data for S. mansoni and Schistosoma japonicum. The large number of probes combined with the extensive sequence annotation available allowed a comprehensive approach, where most of the S. mansoni transcriptome is represented. Hybridization with adult worm RNA pointed to a set of genes transcriptionally active in this stage of the parasite's life cycle. Interestingly, a large proportion (43%) of genes for which transcription was detected in adults is comprised of "no match" genes, i.e. S. mansoni genes with unknown function and no identifiable orthologs in GenBank. Moreover, detection of bi-directional transcription for 7% of the active "no match" genes in adults leads us to hypothesize a widespread production of antisense RNA in S. mansoni, with possible regulatory roles.

Correlation spectroscopy of minor fluorescent species: signal purification and distribution analysis.

We are performing experiments that use fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) to monitor the movement of an individual donor-labeled sliding clamp protein molecule along acceptor-labeled DNA. In addition to the FRET signal sought from the sliding clamp-DNA complexes, the detection channel for FRET contains undesirable signal from free sliding clamp and free DNA. When multiple fluorescent species contribute to a correlation signal, it is difficult or impossible to distinguish between contributions from individual species. As a remedy, we introduce "purified FCS", which uses single molecule burst analysis to select a species of interest and extract the correlation signal for further analysis. We show that by expanding the correlation region around a burst, the correlated signal is retained and the functional forms of FCS fitting equations remain valid. We demonstrate the use of purified FCS in experiments with DNA sliding clamps. We also introduce "single-molecule FCS", which obtains diffusion time estimates for each burst using expanded correlation regions. By monitoring the detachment of weakly-bound 30-mer DNA oligomers from a single-stranded DNA plasmid, we show that single-molecule FCS can distinguish between bursts from species that differ by a factor of 5 in diffusion constant.

Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis.

RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal-ion catalysis is a general feature of the superfamily. In nucleases, the two metal ions are asymmetrically coordinated and have distinct roles in activating the nucleophile and stabilizing the transition state. In transposases, they are symmetrically coordinated and exchange roles to alternately activate a water and a 3'-OH for successive strand cleavage and transfer by a ping-pong mechanism.

Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.

We predict regulatory targets of vertebrate microRNAs (miRNAs) by identifying mRNAs with conserved complementarity to the seed (nucleotides 2-7) of the miRNA. An overrepresentation of conserved adenosines flanking the seed complementary sites in mRNAs indicates that primary sequence determinants can supplement base pairing to specify miRNA target recognition. In a four-genome analysis of 3' UTRs, approximately 13,000 regulatory relationships were detected above the estimate of false-positive predictions, thereby implicating as miRNA targets more than 5300 human genes, which represented 30% of our gene set. Targeting was also detected in open reading frames. In sum, well over one third of human genes appear to be conserved miRNA targets.

La revolución de los microarrays en la investigación biosanitaria: tipos de plataformas, usos y perspectivas en oncología / The microarray revolution in biomedical research: types of platforms, uses and perspectives in oncology

Mediante la introducción, uso y manejo de herramientas de base genómica, la investigación sobre las alteraciones genéticas que están en el origen de enfermedades tan comunes como el cáncer han sufrido una revolución técnica comparable a la incorporación del microscopio en los laboratorios. Ahora, estudiar la relación gen-enfermedad no está basado en analizar un gen único y sus efectos sino en analizar el comportamiento de miles de genes de forma simultánea. Estos sistemas, denominados genéricamente como matrices, arrays, microarrays o biochips, están cambiando nuestra forma de plantear problemas y extraer conclusiones de los experimentos ya que nos ofrecen una foto compleja del conjunto del genoma. Los análisis de expresión mediante microarrays de cDNA o de oligos ya son accesibles a la comunidad científica española. Los resultados, además, fascinan a los investigadores ya que son bastante reproducibles y aportan una gran cantidad de información sobre la regulación de la expresión génica en condiciones normales y patológicas (AU)

El diagnóstico genético preimplantacional y sus nuevas indicaciones en reproducción asistida / Pre-implantation genetic diagnosis and the new indications for its use in assisted reproduction

No disponible

Translocaciones cromosómicas en los linfomas no Hodking (Parte I) / Chromosomal translocations in non-Hodking`s lymphomas (Part I)

No disponible

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.

Molecular epidemiology of bovine anaplasmosis

Bovine anaplasmosis presents a worldwide distribution. However, specific models for studying the epidemiology of the disease are not available. Epidemiological modeling encounters some difficulties due to a lack of culturing techniques for Anaplasma marginales, the causative agent, as well as for the lack of typing techniques to characterize strains. The chronic carrier state and the population dynamics of mechanical and biological vector also create difficulties. In addition, conventional serology and blood smear diagnostic techniques fail to detect all chronica carriers. Fortunately the needs for the accurate typing of isolates and for detecting chronica carriers made it possible to encourage the development of new tools based on molecular epidemiology principles. A. marginali isolates can now be typed by using panels of monoclonal antibodies, and the genes coding for some major surface proteins can be expressed or analyzed by looking at the nucleotide arrangement level. In the same manner, the latest techniques for detecting A. marginale chronic infections use DNA and RNA probes, and PCR-based methods to detect A. marginali DNA from bovine blood samples with extremely low rickettsaemias. Currently all these new epidemiological tools are being incroporated to experimental models to analyze their applicability for epidemiological studies in the near future

RecA protein promotes rapid RNA-DNA hybridization in heterogeneous RNA mixtures.

The nucleoprotein filament formed by the RecA protein of Escherichia coli on single-stranded DNA catalyzes the hybridization of RNA transcripts with single-stranded DNA sequences at 37 degrees C, in vitro. RecA protein rapidly promotes hybridization, even when noncomplementary RNA is in a millionfold nucleotide excess over hybridizing RNA, and in a thousandfold nucleotide excess over hybridizing single-stranded DNA. Heterologous double-stranded DNA and RecA-coated noncomplementary single-stranded DNA are also poor competitors of RNA transcripts produced in vitro. Since large excesses of noncomplementary RNA fail to inhibit sharply the hybridization reaction by RecA protein under mild, non-degradative conditions, the reaction may be useful in the identification and isolation of transcripts produced in vivo.

RNA-DNA hybridization promoted by E. coli RecA protein.

RecA protein of E. coli plays a central regulatory role that is induced by damage to DNA and results in the inactivation of LexA repressor. In vitro, RecA protein binds preferentially to single-stranded DNA to form a nucleoprotein filament that can recognize homology in naked duplex DNA and promote extensive strand exchange. Although RecA protein shows little tendency at neutral pH to bind to RNA, we found that it nonetheless catalyzed at 37 degrees C the hybridization of complementary RNA and single-stranded DNA sequences. Hybrids made by RecA protein at 37 degrees C appeared indistinguishable from ones prepared by thermal annealing. RNA-DNA hybridization by RecA protein at neutral pH required, as does RecA-promoted homologous pairing, optimal conditions for the formation of RecA nucleoprotein filaments. The cosedimentation of RNA with those filaments further paralleled observations made on the formation of networks of nucleoprotein filaments with double-stranded DNA, an instrumental intermediate in homologous pairing in vitro. These similarities with the pairing reaction support the view that RecA protein acts specifically in the hybridization reaction.