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Cytosine methylation is a DNA modification generally associated with transcriptional silencing. Factors that regulate methylation have been linked to human disease, yet how they contribute to malignances remains largely unknown. Genomic maps of DNA methylation have revealed unexpected dynamics at gene regulatory regions, including active demethylation by TET proteins at binding sites for transcription factors. These observations indicate that the underlying DNA sequence largely accounts for local patterns of methylation. As a result, this mark is highly informative when studying gene regulation in normal and diseased cells, and it can potentially function as a biomarker. Although these findings challenge the view that methylation is generally instructive for gene silencing, several open questions remain, including how methylation is targeted and recognized and in what context it affects genome readout., Methylation of nucleotides provides a molecular means to reversibly mark genomic DNA. Bacteria can methylate adenosine or cytosine to identify and degrade invading DNA and to track mismatch repair and [...]

Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring ~60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA--protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA--protein cross-links and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by ~4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA-protein cross-links and generate double strand breaks, the most deleterious form of DNA damage. chromatin | oxidative damage | histone modification doi/ 10.1073/pnas.1012860108

Biomolecular logic devices can be applied for sensing and nanomedicine. We built three DNA tweezers that are activated by the inputs [H.sup.+]/O[H.sup.-]; [Hg.sup.2+]/cysteine; nucleic acid linker/complementary antilinker to yield a 16-states finite-state automaton. The outputs of the automata are the configuration of the respective tweezers (opened or closed) determined by observing fluorescence from a fluorophore/quencher pair at the end of the arms of the tweezers. The system exhibits a memory because each current state and output depend not only on the source configuration but also on past states and inputs. biocomputing | DNA machines | sensor | chemical recognition | chemical input doi/ 10.1073/pnas.1015858107

Understanding and manipulation of the forces assembling DNA/ RNA helices have broad implications for biology, medicine, and physics. One subject of significance is the attractive force between dsDNA mediated by polycations of valence [greater than or equal to] 3. Despite extensive studies, the physical origin of the 'like-charge attraction' remains unsettled among competing theories. Here we show that triple-strand DNA (tsDNA), a more highly charged helix than dsDNA, is precipitated by alkaline-earth divalent cations that are unable to condense dsDNA. We further show that our observation is general by examining several cations ([Mg.sup.2+], [Ba.sup.2+], and [Ca.sup.2+]) and two distinct tsDNA constructs. Cation-condensed tsDNA forms ordered hexagonal arrays that redissolve upon adding monovalent salts. Forces between tsDNA helices, measured by osmotic stress, follow the form of hydration forces observed with condensed dsDNA. Probing a well-defined system of point-like cations and tsDNAs with more evenly spaced helical charges, the counterintuitive observation that the more highly charged tsDNA (vs. dsDNA) is condensed by cations of lower valence provides new insights into theories of polyelectrolytes and the biological and pathological roles of tsDNA. Cations and tsDNAs also hold promise as a model system for future studies of DNA--DNA interactions and electrostatic interactions in general. DNA condensation | small angle X-ray diffraction doi/ 10.1073/pnas.1003374107

The structure and integrity of DNA is of considerable biological and biomedical importance, and it is therefore critical to identify and to characterize enzymes that alter DNA structure. DNA helicases are ATP-driven motor proteins that unwind DNA. Conversely, HepA-related protein (HARP) protein (also known as SMARCAL1 and DNA-dependent ATPase A) is an annealing helicase that rewinds DNA in an ATP-dependent manner. To date, HARP is the only known annealing helicase. Here we report the identification of a second annealing helicase, which we term AH2, for annealing helicase 2. Like HARP, AH2 catalyzes the ATP-dependent rewinding of replication protein A (RPA)-bound complementary single-stranded DNA, but does not exhibit any detectable helicase activity. Unlike HARP, however, AH2 lacks a conserved RPA-binding domain and does not interact with RPA. In addition, AH2 contains an HNH motif, which is commonly found in bacteria and fungi and is often associated with nuclease activity. AH2 appears to be the only vertebrate protein with an HNH motif. Contrary to expectations, purified AH2 does not exhibit nuclease activity, but it remains possible that AH2 contains a latent nuclease that is activated under specific conditions. These structural and functional differences between AH2 and HARP suggest that different annealing helicases have distinct functions in the cell. SNF2 family protein | chromatin | DNA repair | genome integrity doi/ 10.1073/pnas.1011196107

Coarse-grained (CG) modeling approaches are widely used to simulate many important biological processes involving DNA, including chromatin folding and genomic packaging. The bending propensity of a semiflexible DNA molecule critically influences these processes. However, existing CG DNA models do not retain a sufficient fidelity of the important local chain motions, whose propagation at larger length scales would generate correct DNA persistent lengths, in particular when the solution's ionic strength is widely varied. Here we report on a development of an accurate CG model for the double-stranded DNA chain, with explicit treatment of mobile ions, derived systematically from all-atom molecular dynamics simulations. Our model generates complex local motions of the DNA chain, similar to fully atomistic dynamics, leading also to a quantitative agreement of our simulation results with the experimental data on the dependence of the DNA persistence length on the solution ionic strength. We also predict a structural transition in a torsionally stressed DNA nanocircle as the buffer ionic strength is increased beyond a threshold value. molecular simulations | polymer chain | one-step renormalization | chemical coarse graining | explicit ion electrostatics doi/ 10.1073/pnas.1001163107

MutL[alpha] (MLH1-PMS2) is a latent endonuclease that is activated in a mismatch-, MutS[alpha]-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutL[alpha] activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutL[alpha] function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutL[alpha] incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion. MutLalpha | genetic instability | cancer | triplet repeats www.pnas.org/cgi/doi/10.1073/pnas.1010662107

Optofluidic dye lasers hold great promise for adaptive photonic devices, compact and wavelength-tunable light sources, and micro total analysis systems. To date, however, nearly all those lasers are directly excited by tuning the pump laser into the gain medium absorption band. Here we demonstrate bioinspired optofluidic dye lasers excited by FRET, in which the donor-acceptor distance, ratio, and spatial configuration can be precisely controlled by DNA scaffolds. The characteristics of the FRET lasers such as spectrum, threshold, and energy conversion efficiency are reported. Through DNA scaffolds, nearly 100% energy transfer can be maintained regardless of the donor and acceptor concentration. As a result, efficient FRET lasing is achieved at an unusually low acceptor concentration of micromolar, over 1,000 times lower than that in conventional optofluidic dye lasers. The lasing threshold is on the order of [micro]/[mm.sup.2]. Various DNA scaffold FRET lasers are demonstrated to illustrate vast possibilities in optofluidic laser designs. Our work opens a door to many researches and applications such as intracavity bio/chemical sensing, biocontrolled photonic devices, and biophysics. fluorescence resonance energy transfer | optofluidics | ring resonators www.pnas.org/cgi/doi/10.1073/pnas.1003581107

Previous work showed that about 85% of stress-induced mutations associated with DNA double-strand break repair in carbon-starved Escherichia coli result from error-prone DNA polymerase IV (Pol IV) (DinB) and that the mutagenesis is controlled by the RpoS stress response, which upregulates dinB. We report that the remaining mutagenesis requires high-fidelity Pol II, and that this component also requires RpoS. The results identify a second DNA polymerase contributing to stress-induced mutagenesis and show that RpoS promotes mutagenesis by more than the simple upregulation of dinB. doi: 10.1128/JB.00570-10

For many aspects of DNA-protein interaction, it is vital to know how DNA bending rigidity (or persistence length, a) depends on its sequence. We addressed this problem using the method based on cyclization of short DNA fragments, which allows very accurate determination of a. Our approach was based on assigning specific values of a to each of 10 distinct dinucleotide steps. We prepared DNA fragments, each about 200 bp in length, with various quasi-periodic sequences, measured their cyclization efficiencies (j factors), and fitted the data by the theoretical equation to obtain the values of a for each fragment. From these data, we obtained a set of a for the dinucleotide steps. To test this set, we used it to design DNA sequences that should correspond to very low and very high values of a, prepared the corresponding fragments, and determined their values of a experimentally. The measured and calculated values of a were very close to one another, confirming that we have found the correct solution to this long-standing problem. The same experimental data also allowed us to determine the sequence dependence of DNA helical repeat. DNA elasticity | DNA persistence length doi/ 10.1073/pnas.1004809107

Fanconi anemia (FA) is caused by mutations in 13 Font genes and renders ceils hypersensitive to DNA interstrand cross-linking (ICL) agents. A central event in the FA pathway is mono-ubiquitylation of the FANCI-FANCD2 (ID) protein complex. Here, we characterize a previously unrecognized nuclease, Fanconi anemia-associated nuclease 1 (FAN1), that promotes ICL repair in a manner strictly dependent on its ability to accumulate at or near sites of DNA damage and that relies on mono-ubiquitylation of the ID complex. Thus, the mono-ubiquitylated ID complex recruits the downstream repair protein FAN1 and facilitates the repair of DNA interstrand cross-links. 10.1126/science.1192656

We demonstrate the use of both optical extinction and phase measurements for probing the interactions between DNA and small molecules by dual polarization interferometry. On binding to DNA at the interface, mitoxantrone (MTX) and methylene blue (MB) induced reversible concentration-dependent optical extinction due to light absorption, which clearly revealed the association end dissociation of small molecules with DNA in real time. The binding constants of MTX--DNA and MB--DNA determined from the masses derived from optical extinction are 1.8 x [10.sup.5] and 4.2 x [10.sup.4] [M.sup.-1], respectively, and shown to be buffer salt concentration-dependent. Apart from optical extinction, phase measurements reflected the overall change of the interaction; namely, a combined result of the binding of small molecules and any changes in DNA structure. The masses derived from phase could be very different from those derived from optical extinction. The structural changes detected by phase measurements showed a contraction and densification of DNA upon intercalation by MTX or MB. The combination of optical extinction and phase measurements allows a detailed understanding of the interaction process. 10.1021/ac9027164

A single-stranded DNA-binding protein (SSB) has been recently proven to facilitate highly efficient separation of the excess hybridization probe from the probe-target hybrid in gel-free capillary electrophoresis. SSB added to the electrophoresis run buffer binds the single-stranded DNA probe but does not bind the double stranded DNA--DNA or DNA--RNA hybrid. As a result, SSB changes the electrophoretic mobility of the probe but does not affect the mobility of the hybrid. If the probe is labeled fluorescently, real-time sensitive detection can be facilitated. While the concept of SSB-mediated hybridization analysis has been proven in principle, the question of how to make such analysis quantitative without building a calibration curve remains open. Here, we propose a general approach for making SSB-mediated analysis quantitative. This approach takes into account such phenomena as (i) the potential influence of the probe--target hybridization and probe--SSB binding on the quantum yield of the fluorescent label and (ii) the potential dissociation of the hybrid by SSB. The proposed approach was used to study the quenching and the dissociation phenomena experimentally. We proved for the first time that SSB does not detectably dissociate the probe--target hybrid, which significantly simplifies the analysis. 10.1021/ac100537p

A general methodology to design label-free fluorescent functional DNA sensors using unmodified DNA via a vacant site approach is described. By extending one end of DNA with a loop, a vacant site that binds an extrinsic fluorophore, 2-amino- 5,6,7-trimethyl-1,8-naphthyridine (ATMND), could be created at a selected position in the DNA duplex region of DNAzymes or aptamers. When the vacant site binds ATMND, ATMND's fluorescence is quenched. This fluorescence can be recovered when one strand of the duplex DNA is released through either metal ion-dependent cleavage by DNAzymes or analyte-dependent structural-switching by aptamers. Through this design, label-free fluorescent sensors for [Pb.sup.2+], U[O.sub.2.sup.2+], [Hg.sup.2], and adenosine have been successfully developed. These sensors have high selectivity and sensitivity; detection limits as low as 3 nM, 8 nM, 30 nM, and 6 [micro]M have been achieved for U[O.sub.2.sup.2+], [Pb.sup.2+], [Hg.sup.2+] and adenosine, respectively. Control experiments using vacant-site-free DNA duplexes and inactive variants of the functional DNAs indicate that the presence of the vacant site and the activity of the functional DNAs are essential for the performance of the proposed sensors. The vacant site approach demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes. 10.1021/ac100244h

Eukaryotic cytosine methylation represses transcription but also occurs in the bodies of active genes, and the extent of methylation biology conservation is unclear. We quantified DNA methylation in 17 eukaryotic genomes and found that gene body methylation is conserved between plants and animals, whereas selective methylation of transposons is not. We show that methylation of plant transposons in the CHG context extends to green algae and that exclusion of histone H2A.Z from methylated DNA is conserved between plants and animals, and we present evidence for RNA-directed DNA methylation of fungal genes. Our data demonstrate that extant DNA methylation systems are mosaics of conserved and derived features, and indicate that gene body methylation is an ancient property of eukaryotic genomes. 10.1126/science.1186366

The centrosomal protein, CDK5RAP2, is mutated in primary microcephaly, a neurodevelopmental disorder characterized by reduced brain size. The Drosophila melanogaster homologue of CDK5RAP2, centrosomin (Cnn), maintains the pericentriolar matrix (PCM) around centrioles during mitosis. In this study, we demonstrate a similar role for CDK5RAP2 in vertebrate cells. By disrupting two evolutionarily conserved domains of CDK5RAP2, CNN1 and CNN2, in the avian B cell line DT40, we find that both domains are essential for linking centrosomes to mitotic spindle poles. Although structurally intact, centrosomes lacking the CNN1 domain fail to recruit specific PCM components that mediate attachment to spindle poles. Furthermore, we show that the CNN1 domain enforces cohesion between parental centrioles during interphase and promotes efficient DNA damage--induced G2 cell cycle arrest. Because mitotic spindle positioning, asymmetric centrosome inheritance, and DNA damage signaling have all been implicated in cell fate determination during neurogenesis, our findings provide novel insight into how impaired CDK5RAP2 function could cause premature depletion of neural stem cells and thereby microcephaly. doi/10.1083/jcb.200912163

Left-handed Z-DNA has fascinated biological scientists for decades by its extraordinary structure and potential involvement in biological phenomena. Despite its instability relative to B-DNA, Z-DNA is stabilized in vivo by negative supercoiling. A detailed understanding of Z-DNA formation is, however, still lacking. In this study, we have examined the B-Z transition in a short guanine/cytosine (GC) repeat in the presence of controlled tension and superhelicity via a hybrid technique of single-molecule FRET and magnetic tweezers. The hybrid scheme enabled us to identify the states of the specific GC region under mechanical control and trace conformational changes synchronously at local and global scales. Intriguingly, minute negative superhelicity can facilitate the B-Z transition at low tension, indicating that tension, as well as torsion, plays a pivotal role in the transition. Dynamic interconversions between the states at elevated temperatures yielded thermodynamic and kinetic constants of the transition. Our single-molecule studies shed light on the understanding of Z-DNA formation by highlighting the highly cooperative and dynamic nature of the B-Z transition. magnetic tweezers | single-molecule FRET | Z-DNA doi/ 10.1073/pnas.0911528107

Probe design is a critical parameter in successful DNA and RNA target detection. In this proof-of-concept study, we evaluated the single-base mismatch recognition power of surface immobilized and self-assembled stem-loop hairpin DNA oligonucleotide probes modified to contain locked nucleic acid residues (LNA-HP). The stiffness change in conjunction with the stem opening of the interfacial molecules before and after hybridization led to clear variations of the overall film thickness or miniaturized nanospot height, which could be directly measured using an atomic force microscopy (AFM) nanolithography technique. Particularly, LNA-HP achieved highly differentiable readouts between perfectly complementary and singly mismatched targets (discrimination ratio as high as 2 to 3), outperforming the selectivity of its linear and hairpin counterparts with no LNA modification. 10.1021/ac902665c

Histones play a crucial role in the organization of DNA in the nucleus, but their presence can prevent interactions with DNA binding proteins responsible for repair of DNA damage. Uracil is an abundant mutagenic lesion recognized by uracil DNA glycosylase (UDG) in the first step of base excision repair (BER). In nucleosome core particles (NCPs), we find substantial differences in UDG-directed cleavage at uracils rotationally positioned toward (U-In) or away from (U-Out) the histone core, or midway between these orientations (U-Mid). Whereas U-Out NCPs show a cleavage rate just below that of naked DNA, U-In and U-Mid NCPs have markedly slower rates of cleavage. Crosslinking of U-In DNA to histones in NCPs yields a greater reduction in cleavage rate but, surprisingly, yields a higher rate of cleavage in U-Out NCPs compared with uncrosslinked NCPs. Moreover, the next enzyme in BER, APE1, stimulates the activity of human UDG in U-Out NCPs, suggesting these enzymes interact on the surface of histones in orientations accessible to UDG. These data indicate that the activity of UDG likely requires 'trapping' transiently exposed states arising from the rotational dynamics of DNA on histones. chromatin | DNA damage | glycosylase | histones | APE1 endonuclease www.pnas.org/cgi/doi/10.1073/pnas.0914443107

It is generally assumed that sister chromatids are genetically and functionally identical and that segregation to daughter cells is a random process. However, functional differences between sister chromatids regulate daughter cell fate in yeast (1) and sister chromatid segregation is not random in Escherichia coli (2). Differentiated sister chromatids, coupled with non-random segregation, have been proposed to regulate cell fate during the development of multicellular organisms (3). This hypothesis has not been tested because molecular features to reliably distinguish between sister chromatids are not obvious. Here we show that parental 'Watson' and 'Crick' DNA template strands can be identified in sister chromatids of murine metaphase chromosomes using CO-FISH (chromosome orientation fluorescence in situ hybridization (4)) with unidirectional probes specific for centromeric and telomeric repeats. All chromosomes were found to have a uniform orientation with the 5' end of the short arm on the same strand as T-rich major satellite repeats. The invariable orientation of repetitive DNA was used to differentially label sister chromatids and directly study mitotic segregation patterns in different cell types. Whereas sister chromatids appeared to be randomly distributed between daughter cells in cultured lung fibroblasts and embryonic stem cells, significant non-random sister chromatid segregation was observed in a subset of colon crypt epithelial cells, including cells outside positions reported for colon stem cells (5). Our results establish that DNA template sequences can be used to distinguish sister chromatids and follow their mitotic segregation in vivo., Major satellite repeats have a uniform head-to-tail orientation on mouse chromosomes relative to the centromeri (6,7). To determine whether this polarity is fixed relative to chromosome ends, we hybridized unidirectional [...]

A new strategy for molecular beacon binding readout is proposed by using separation of the molecular recognition element and signal reporter. The signal transduction of the target binding event is based on displacing interaction between the target DNA and a competitor, the signal transducer. The target-free capture DNA is first interacted with the competitor, forming an assembled complex. In the presence of a target DNA that the affinity is stronger than that of the competitor, hybridization between capture DNA and the target disassembles the assembled complex and releases the free competitor to change the readout of the signal reporter. To demonstrate the feasibility of the design, a thymine-rich oligonucleotide was examined as a model system. [Hg.sup.2+] was selected as the competitor, and mercaptoacetic acid-coated CdTe/ZnS quantum dots served as the fluorescent reporter. Selective binding of [Hg.sup.2+] between the two thymine bases of the capture DNA forms a hairpin-structure. Hybridization between the capture DNA and target DNA destroys the hairpin-structure, releasing [Hg.sup.2+] ions to quench the quantum dots fluorescence. Under the optimal conditions, fluorescence intensity of the quantum dots against the concentration of perfect cDNA was linear over the concentration range of 0.1-1.6 [micro]M, with a limit of detection of 25 nM. This new assay method is simple in design, avoiding any oligonucleotide labeling. Furthermore, this strategy is generalizable since any target binding can in principle release the signal transducer and be detected with separated signal reporter. 10.1021/ac901906w

It has been suggested that the structure that results when double-stranded DNA (dsDNA) is pulled from the 3'3' ends differs from that which results when it is pulled from the 5'5' ends. In this work, we demonstrate, using [lambda] phage dsDNA, that the overstretched states do indeed show different properties, suggesting that they correspond to different structures. For 3'3' pulling versus 5'5' pulling, the following differences are observed: (i) the forces at which half of the molecules in the ensemble have made a complete force-induced transition to single stranded DNA are 141 [+ or -] 3 pN and 122 [+ or -] 4 pN, respectively; (ii) the extension vs. force curve for overstretched DNA has a marked change in slope at 127 [+ or -] 3 pN for 3'3' and 110 [+ or -] 3 pN for 5'5'; (iii) the hysteresis (H) in the extension vs. force curves at 150 mM NaCl is 0.3 [+ or -] 0.8 pN [micro]m for 3'3' versus 13 [+ or -] 8 pN for 5'5'; and (iv) 3'3' and 5'5' molecules show different changes in hysteresis due to interactions with [beta]-cyclodextrin, a molecule that is known to form stable host-guest complexes with rotated base pairs, and glyoxal that is known to bind stably to unpaired bases. These differences and additional findings are well-accommodated by the corresponding structures predicted on theoretical grounds.

We present an analysis of the key steps involved in the DNA-directed assembly of nanoparticles into crystallites and polycrystalline aggregates. Additionally, the rate of crystal growth as a function of increased DNA linker length, solution temperature, and self-complementary versus non-self-complementary DNA linker strands (1- versus 2-component systems) has been studied. The data show that the crystals grow via a 3-step process: an initial 'random binding' phase resulting in disordered DNA-AuNP aggregates, followed by localized reorganization and subsequent growth of crystalline domain size, where the resulting crystals are well-ordered at all subsequent stages of growth. DNA materials | SAXS | self assembly

The electrochemical behavior of a trinuclear ruthenium(II)-containing complex, [[[([(phen).sub.2]Ru(dpp)).sub.2]Rh[Cl.sub.2]].sup.5+] (where phen = 1,10-phenanthroline, dpp = 2,3-bis(2-pyridyl)pyrazine), was studied in acetonitrile (MeCN) and aqueous solutions. In MeCN containing 0.10 M tetran-butylammonium perchlorate (TBAP), the complex displayed a reversible, overlapping [Ru.sup.II/III] redox process with [E.sub.1/2] = +1.21 V vs Ag/[Ag.sup.+] (10 mM), an irreversible reduction of [Rh.sup.III/I] at -0.73 V vs Ag/[Ag.sup.+], and two quasi-reversible dpp/[dpp.sup.-] couples with [E.sub.1/2] = -1.11 and -1.36 V vs Ag/[Ag.sup.+] at a Pt electrode with a scan rate of 50 mV [s.sup.-1]. In 0.20 M Tris buffer solution (pH 7.4), an irreversible, overlapping [Ru.sup.II/III] oxidation at +1.48 V vs Ag/AgCl (3 M KC1), and an irreversible reduction of [Rh.sup.III/II] at -0.78 V vs Ag/AgCl were observed at a glassy carbon electrode with a scan rate of 50 mV/s. Investigations on the electrogenerated chemiluminescence (ECL) of the complex revealed that 2-(dibutylamino) ethanol (DBAE) was superior to trinpropylamine (TPrA) as an ECL coreactant within their entire concentration range of 10-100 mM in MeCN, and in aqueous media, as low as 1.0 nM of the complex can be detected using TPrA coreactant ECL. A maximum ECL emission of 640 nm, which is about 55 nm blue shift to its fluorescence, was observed in MeCN with DBAE as a coreactant. Interactions of the complex with calf thymus DNA (ctDNA) were conducted with a flow-cell based quartz-crystal microbalance, and a binding constant of 2.5 x [10.sup.5] [M.sup.-1] was calculated on the basis of the Langmuir isotherm equation.

In our previous work, we selected aptamers binding to ethanolamine, one of the smallest molecular aptamer targets so far (Mann, D.; Reinemann, C.; Stoltenburg, R.; Strehlitz, B. Biochem. Biophys. Res. Commun. 2005, 338, 1928-1934). Two representatives of these aptamers (EA#14.3 and EA#9.4) were analyzed regarding their specificity. Ethanolamine is a very small organic molecule ([M.sub.w] = 61.08) with biological, medical, and industrial relevance. Its small size represented a challenge for aptamer development, as ethanolamine only consists of a short carbon chain (2C) and two functional groups (amino and hydroxyl group). Related organic molecules, ethanolamine derivatives, and some amino acids were tested to act as potential binding partners for these aptamers. In this way we were able to determine the exact binding domain within the target. The results revealed that both aptamers bind to various molecules, which contain a freely accessible ethyl- or methylamine group. In contrast to the amino group (in a primary, secondary, or tertiary amine) the hydroxyl group was not necessary for the aptamer binding. The aptamers were not able to bind to negatively charged organic molecules, despite containing an ethyl- or methylamine group, nor did they bind to molecules with quaternary amines. The selected ethanolamine binding aptamers are useful for the detection of molecules containing accessible ethyl- or methylamine groups; they can be used as linker elements to immobilize a target molecule of interest on a surface or to purify targets from complex samples.

We report for the first time that peptide could serve as a medium to bring an anionic conjugated polymer and a dye-labeled DNA into close proximity for energy transfer. By taking advantage of the fluorescein (F1)-labeled peptide-mediated energy transfer between poly(9,9-bis(4'-sulfonatobutyl)fluorene-alt-1,4-phenylene) sodium salt (PFP-S[O.sub.3]Na) and Texas red (TR)-labeled single-stranded DNA (ssDNA), we develop a homogeneous assay for detection and monitoring of protease and nuclease activity in one solution using peptide/DNA complexes as the substrate. The enzymes as a proof of concept are trypsin (protease) and S1 (nuclease), respectively. In the absence of enzyme, multistep fluorescence energy transfer occurs from PFP-S[O.sub.3]Na to F1 and TR and from F1 to TR, and the TR emission dominates the solution fluorescence. In the presence of trypsin, the peptide is cleaved into fragments; the relatively weak electrostatic interaction between the small peptide fragments and the polymer fails to bring the TR-ssDNA and the polymer into close proximity for energy transfer. There is a significant decrease in TR emission and an increase in PFP-S[O.sub.3]Na emission, and the solution fluorescence appears blue. When S1 nuclease is used to cleave TR-ssDNA, a significant decrease in TR emission and an obvious increase in F1 emission are found, and the solution fluorescence appears green. The developed assay is ideal for the detection of chemical and biological molecules with DNA or protein cleaving activities.

RNA interference pathways use small RNAs to mediate gene silencing in eukaryotes. In addition to small interfering RNAs (siRNAs) and microRNAs, several types of endogenously produced small RNAs have important roles in gene regulation, germ cell maintenance and transposon silencing (1-4). The production of some of these RNAs requires the synthesis of aberrant RNAs (aRNAs) or pre-siRNAs, which are specifically recognized by RNA-dependent RNA polymerases to make double-stranded RNA. The mechanism for aRNA synthesis and recognition is largely unknown. Here we show that DNA damage induces the expression of the Argonaute protein QDE-2 and a new class of small RNAs in the filamentous fungus Neurospora crassa. This class of small RNAs, known as giRNAs because of their interaction with QDE-2, are about 20-21 nucleotides long (several nucleotides shorter than Neurospora siRNAs), with a strong preference for uridine at the 5' end, and originate mostly from the ribosomal DNA locus. The production of giRNAs requires the RNA-dependent RNA polymerase QDE-1, the Werner and Bloom RecQ DNA helicase homologue QDE-3 and dicers. giRNA biogenesis also requires DNA-damage-induced aRNAs as precursors, a process that is dependent on both QDE1 and QDE-3. Notably, our results suggest that QDE-1 is the DNA-dependent RNA polymerase that produces aRNAs. Furthermore, the Neurospora RNA interference mutants show increased sensitivity to DNA damage, suggesting a role for giRNAs in the DNA-damage response by inhibiting protein translation., In the filamentous fungus Neurospora crassa, the RNA interference (RNAi) pathway is essential for both double-stranded RNA (dsRNA)-induced and transgene-induced gene silencing (quelling) (5). In the quelling pathway, QDE-1 and [...]

We have developed a new fluorescent ensemble probe comprising an ionic conjugate between water-soluble thioglycolic acid (TGA) capped CdTe quantum dots (QDs) and [Ru(bpy).sub.2][(dppx).sup.2+] for the dual-color detection of complementary double-stranded DNAs (dsDNA). To provide the platform for DNA detection, the Rucomplex was first employed as an effective fluorescence quencher to TGA capped QDs via photoinduced electron transfer process. Because of its strong binding affinity with [Ru(bpy).sub.2][(dppx).sup.2+], complementary dsDNA can break up the low fluoresced ionic ensemble, set free the luminescent QDs, and concomitantly generate the [Ru(bpy).sub.2][(dppx).sup.2+] intercalated DNA complex. Thus, the recognition of dsDNA by [Ru(bpy).sub.2][(dppx).sup.2+] can be realized via both the restoration of QDs fluorescence and the emergence of a new fluorescence emission signal of the quencher--substrate at 609 nm, while single-stranded DNA, ribonucleic acid, bovine albumin serum, and biological relevant metal ions cannot produce the similar results. Therefore, a simple, fast, sensitive, and highly selective assay for dsDNA has been realized.

An automated sample introduction system, utilizing a demountable direct injection high-efficiency nebulizer (d-DIHEN), is successfully incorporated for the first time with an inductively coupled plasma optical emission spectrometer (ICP-OES) for the measurement of the phosphorus content in acid-digested nucleotides and deoxyribonucleic acid (DNA). With this experimental setup, the solution uptake rate and volume are reduced from 170 to 30 [micro]L [min.sup.-1] and from 10 to 2.4 mL, respectively, thereby reducing the required DNA sample mass for solutions containing 3 [micro]g [g.sup.-1] p from 300 to 72 [micro]g of DNA, in comparison to previous analyses in our laboratory using a glass concentric nebulizer with cyclonic spray chamber arrangement. The use of direct injection also improves P (I) 213.617 nm sensitivity by a factor of 4 on average. A high-performance (HP) methodology in combination with the previous sample introduction system and ICP-OES provides simultaneous, time-correlated internal standardization and drift correction resulting in relative expanded uncertainties (% U) for the P mass fractions in the range of 0.1-0.4 (95% confidence level) for most of the thymidine 5'-monophosphate (TMP), calf thymus DNA (CTDNA), and plasmid DNA (PLDNA) analyses. The d-DIHEN with HP-ICP-OES methodology allows for the quantification of DNA mass at P mass fractions as low as 0.5 [micro]g [g.sup.-1], further reducing the required DNA mass to 12 [micro]g, with small uncertainty ([less than or equal to] 0.4%). This successful approach will aid in the development and certification of nucleic acid certified reference materials (CRMs), particularly for these samples that are typically limited in volume.

The three-dimensional molecular structure of DNA, specifically the shape of the backbone and grooves of genomic DNA, can be dramatically affected by nucleotide changes, which can cause differences in protein-binding affinity and phenotype. We developed an algorithm to measure constraint on the basis of similarity of DNA topography among multiple species, using hydroxyl radical cleavage patterns to interrogate the solvent-accessible surface area of DNA. This algorithm found that 12% of bases in the human genome are evolutionarily constrained--double the number detected by nucleotide sequence--based algorithms. Topography-informed constrained regions correlated with functional noncoding elements, including enhancers, better than did regions identified solely on the basis of nucleotide sequence. These results support the idea that the molecular shape of DNA is under selection and can identify evolutionary history.

Without prior signal amplification, small molecules are difficult to detect by current label-free biochip approaches. In the present study, we developed a label-free capture biochip based on the comparative measurement of unbinding forces allowing for direct detection of small-molecule-aptamer interactions. The principle of this assay relies on increased unbinding forces of bipartite aptamers due to complex formation with their cognate ligands. The bipartite aptamers are immobilized on glass support via short DNA duplexes that serve as references to which unbinding forces can be compared. In a simple model system, adenosine is captured from solution by an adenosine-selective aptamer. Linking the molecular chains, each consisting of a short DNA reference duplex and a bipartite aptamer, between glass and a poly(dimethylsiloxane) (PDMS) surface and subsequently separating the surfaces compares the unbinding forces of the two bonds directly. Fluorescence readout allows for quantification of the fractions of broken aptamer and broken reference bonds. The presence of micromolar adenosine concentrations reliably resulted in a shift toward larger fractions of broken reference bonds. Because of the force-based design, the interactions between the bipartite aptamer and the target, rather than the presence of the target, are detected and no washing step disturbing the equilibrium state prior to probing and no reporter aptamer or antibody is required. The assay exhibits excellent selectivity against other nucleotides and detects adenosine in the presence of a complex molecular background. Multiplexing was demonstrated by performing whole titration experiments on a single chip revealing an effective half-maximal concentration of 124.8 [micro]M agreeing well with literature values.

In electrokinetic trapping (EKT), the electroosmotic velocity of a buffer solution in one area of a microfluidic device opposes the electrophoretic velocity of the analyte in a second area, resulting in transport of DNA to a location where the electrophoretic and electroosmotic velocities are equal and opposite and DNA concentrates at charged nanochannels. The method does not require an optical plug localization, a considerable advantage as compared to preconcentration techniques previously presented. In the work reported here, the trapping process is preceded by a field-amplification in the sample reservoir to reduce trapping time, as field-amplified EKT is shown to be an effective technique to preconcentrate samples from larger volumes. A theoretical model explaining the principle of field-amplified EKT considers different ionic strengths and cross-sectional areas in the microchip segments. The model is supported by experimental data using nucleic acids and fluorescein as sample analytes. An incorporated poly(ethylene terephthalate) (PET) membrane provides anion exclusion due to a negatively charged surface. A fluidic counter flow supports the trapping process in 100 nm pores due to anion exclusion. An analysis of Joule heating gives evidence that temperature gradient focusing effects are negligible and charge exclusion is responsible for trapping. The theoretical model developed and experimentally demonstrated can be exploited for the preconcentration of cell free fetal DNA circulating in maternal plasma and other rare nucleic acid species present in large sample volumes.

Chromatin immunoprecipitation (ChIP) is a powerful and widely applied technique for detecting association of individual proteins with specific genomic regions; the technique requires several complex steps and is tedious. In this paper, we develop a microbead-packed microfluidic chip which eliminates most of the laborious, time-consuming, and skill-dependent processes of the ChIP procedure. A computational fluid dynamics model was established to analyze fluidic behavior in a microbead-packed microchannel. With the use of the new chip, a ChIP procedure was performed to purify the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene from human embryonic kidney cells (cell line 293). The ChIP capability of the microfluidic chip was evaluated and compared with that of a commercial assay kit; the precipitation performance of both methods was almost identical as shown by quantitative measurement of DNA. However, our chip offers the advantage of low resin volume, and the experimental time is greatly reduced. In addition, our method is less dependent on individual technical skill. Therefore, we expect that our microfluidic chip-based ChIP method will be widely used in DNA-, gene-, and protein-related research and will improve experimental efficiency.

Noncoding RNAs (ncRNAs) are now recognized as transregulators of eukaryotic transcription, a role once attributed exclusively to protein factors. Two ncRNAs in mammalian cells have been shown to repress general mRNA transcription by RNA polymerase II (Pol II) in response to heat shock: mouse B2 RNA and human Alu RNA. B2 and Alu RNAs bind directly and tightly to Pol II and co-occupy the promoters of repressed genes along with the polymerase. Here, we identified the molecular mechanism by which mouse B2 RNA and human Alu RNA repress Pol II transcription. Biochemical assays to probe the network of protein--DNA interactions at the promoter revealed that B2 and Alu RNAs prevent Pol II from establishing contacts with the promoter both upstream and downstream of the TATA box during closed complex formation. Disruption of these contacts correlates with transcriptional repression. We conclude that B2 and Alu RNA prevent Pol II from properly engaging the DNA during closed complex formation, resulting in complexes with an altered conformation that are transcriptionally inert. In the absence of its normal contacts with the promoter, Pol II is likely held in these inactive complexes on DNA through interactions with promoter-bound TATA box-binding protein and transcription factor lIB. noncoding RNA | closed complex | open complex

We present an investigation of water menisci confined in closed geometries by studying the structural effects of their capillary forces on viruses during the final stage of desiccation. We used individual particles of the bacteriophage [phi] 29 and the minute virus of mice. In both cases the genomic DNA was ejected from the capsid. However, although the structural integrity of the minute virus of mice was essentially preserved, the [phi] 29 capsid underwent a wall-to-wall collapse. We provide evidence that the capillary forces of water confined inside the viruses are mainly responsible for these effects. Moreover, by performing theoretical simulations with a lattice gas model, we found that some structural differences between these 2 viruses may be crucial to explain the different ways in which they are affected by water menisci forces confined at the nanoscale. atomic force microscopy | capillary forces | water | DNA ejection | virus

A substantial challenge in engineering molecular motors is designing mechanisms to coordinate the motion between multiple domains of the motor so as to bias random thermal motion. For bipedal motors, this challenge takes the form of coordinating the movement of the biped's legs so that they can move in a synchronized fashion. To address this problem, we have constructed an autonomous DNA bipedal walker that coordinates the action of its two legs by cyclically catalyzing the hybridization of metastable DNA fuel strands. This process leads to a chemically ratcheted walk along a directionally polar DNA track. By covalently cross-linking aliquots of the walker to its track in successive walking states, we demonstrate that this Brownian motor can complete a full walking cycle on a track whose length could be extended for longer walks. We believe that this study helps to uncover principles behind the design of unidirectional devices that can function without intervention. This device should be able to fulfill roles that entail the performance of useful mechanical work on the nanometer scale.

A novel fluorescent CdS-encapsulated DNA nanocomposite was synthesized via alternate adsorption of [Cd.sup.2+] and [S.sup.2-] onto the DNA template affixed inside an agarose gel. Confining DNA molecules in the gel matrix reduces the flexibility of the DNA strand, which facilitates the formation of a uniform coating of CdS onto the DNA template. The resultant rod-shaped nanocomposite (40-90 nm in width and 200-300 nm in length) is well dispersed in solution and fluoresces at 330 nm upon excitation at either 228 or 280 nm. The fluorescence is attributed to tiny particles present in the CdS coating. It was found that the fluorescence can be significantly quenched by trace amount of [Hg.sup.2+]. The high selectivity toward [Hg.sup.2+] and the apparent change in the CdS coating upon exposure to [Hg.sup.2+] indicate that [Hg.sup.2+] has reacted with the CdS coating through formation of the much more insoluble HgS and the bridging S--Hg--S bonds at the surface. The extent of quenching is dependent on the concentration of [Hg.sup.2+] in the range of 0.04-13 [micro]M, and a remarkable detection limit (8.6 nM at 30[degrees]C and 4.3 nM at 50[degrees]C) can be achieved. The feasibility of the method for the analysis of [Hg.sup.2+] in a wastewater sample was demonstrated with an excellent relative standard deviation (RSD, 3.4%). The method described herein is simple, selective, and sensitive and obviates the need of extensive sample pretreatment or special instrumentation.

A particle-based multiplexed DNA assay based on encoded porous Si[O.sub.2] photonic crystal disks is demonstrated. A 'spectral barcode' is generated by electrochemical etch of a single-crystal silicon wafer using a programmed current-time waveform. A lithographic procedure is used to isolate cylindrical microparticles 25 [micro]m in diameter and 10 [micro]m thick, which are then oxidized, modified with a silane linker, and conjugated to various amino-functionalized oligonucleotide probes via cyanuric chloride. It is shown that the particles can be decoded based on their reflectivity spectra and that a multiple analyte assay can be performed in a single sample with a modified fluorescence microscope. The homogeneity of the reflectivity and fluorescence spectra, both within and across the microparticles, is also reported.

Enterohemorrhagic Escherichia coli o157, a verocytotoxin (VT1/2)-producing pathogen, can be deadly because it can induce acute or chronic renal failure. To speed up the clinical diagnosis of related syndromes caused by E. coli O157, there is an urgent need for rapid, simple, and reliable analytical tools for its quantitation. In this study, we developed a novel electrochemical competitive genosensor, featuring gold-electrodeposited screen-printed electrodes (nanoAu/SPE) modified with a self-assembled monolayer of thiol-capped single-stranded DNA (capture probe), for the detection of the rfbE gene, which is specific to E. coli O157. This assay functions based on competition between the target gene (complementary to the capture probe DNA) and reporter DNA-tagged, hexaammineruthenium(III) chloride-encapsulated liposomes. The current signal of the released liposomal [Ru(N[H.sub.3]).sub.6.sup.3+] was measured using square wave voltammetry, yielding a sigmoidally shaped dose--response curve whose linear portion was over the range from 1 to [10.sup.6] fmol. This liposomal competitive assay provides an amplification route for the detection of the rfbE gene at ultratrace levels; indeed, we could detect as little as 0.75 amol of the target rfbE DNA (equivalent to the amount present in 5 [micro]L of a 0.15 pM solution).

Mutual recognition of homologous sequences of DNA before strand exchange is considered to be the most puzzling stage of recombination of genes. In 2001, a mechanism was suggested for a double-stranded DNA molecule to recognize from a distance its homologous match in electrolytic solution without unzipping [Kornyshev AA, Leikin S (2001) Phys Rev Lett 86:3666-3669]. Based on a theory of electrostatic interactions between helical molecules, the difference in the electrostatic interaction energy between homologous duplexes and between nonhomologous duplexes, called the recognition energy, was calculated. Here, we report a theoretical investigation of the form of the potential well that DNA molecules may feel sliding along each other. This well, the bottom of which is determined by the recognition energy, leads to trapping of the molecular tracks of the same homology in direct juxtaposition. A simple formula for the shape of the well is obtained. The well is quasi-exponential. Its half-width is determined by the helical coherence length, introduced first in the same 2001 article, the value of which, as the latest study shows, is [approximately equal to] 10 nm.

DNA methylation is essential for silencing transposable elements and some genes in higher eukaryotes, which suggests that this modification must be tightly controlled. However, accidental changes in DNA methylation can be transmitted through mitosis (as in cancer) or meiosis, leading to epiallelic variation. We demonstrated the existence of an efficient mechanism that protects against transgenerational loss of DNA methylation in Arabidopsis. Remethylation is specific to the subset of heavily methylated repeats that are targeted by the RNA interference (RNAi) machinery. This process does not spread into flanking regions, is usually progressive over several generations, and faithfully restores wild-type methylation over target sequences in an RNAi-dependent manner. Our findings suggest an important rote for RNAi in protecting genomes against long-term epigenetic defects.

Arrays of small nanomechanical resonators with dual geometry have been fabricated for sensitive biological detection. The arrays consist of silicon nitride resonating 100 nm thick cantilevers with sensing gold areas alternately placed on the free and fixed cantilever ends. The Au areas act as sensing regions as can be functionalized by means of thiol chemistry. The nanomechanical arrays provide a double flavor of the adsorbed molecules: the added mass reported by the cantilevers with the Au area at the tip and the nanoscale elasticity reported by the cantilevers with the Au area at the clamp. The devices were applied for DNA detection based on Watson-Crick pairing rules. The proposed design for nanomechanical resonators provides higher specificity for DNA sensing in comparison with conventional single cantilevers. The nanoscale elasticity induced by the DNA hybridization arises from the intermolecular interactions between the adsorbates bound to the cantilever and the surface stress.

The study of dynamics and thermodynamics of single biological molecules at confined liquid-solid interfaces is crucially important, especially in the case of low-copy number molecules in a single cell. Using a high-throughput single molecule imaging system and Lagrangian coordinates of single molecule images, we discovered that the local equilibrium isotherms of single [lambda]DNA molecules at a confined liquid-solid interface varied from a stair type for the regions of single or double molecular DNA to a mild 'S' type for the regions of triple molecular DNA spots, which does not agree with the conventional equilibrium isotherms in the literature. Single molecule images in time sequence for different [lambda]DNA concentrations were statistically analyzed by measuring preferential partitioning from shearing effects, which were used to measure the local velocity of DNA molecules by directly observing the migration of DNA fluorescence spots for the 12 continuous images. The local linear velocity of hydrodynamic flow was calculated by the Hagen-Poiseuille equation in different microregions with a local Lagrangian approach. The local single molecule isotherms for the tracked molecules in the regions of single, double, or triple molecular DNA layers within the laminar flows were obtained according to the average local velocities of both the stochastic molecule events and the corresponding local Poiseuille flows. A millisecond and microvolume approach to directly determine local single molecule isotherms at confined liquid-solid interfaces was established, and the microspace scale effects on the types of isotherms were discovered. This study may have significant impact on preparations of low-copy number proteins in a single cell, membrane separations, and other bioseparation studies.

In many settings, molecular testing is needed but unavailable due to complexity and cost. Simple, rapid, and specific DNA detection technologies would provide important alternatives to existing detection methods. Here we report a novel, rapid nucleic acid detection method based on the accelerated photobleaching of the light-sensitive cyanine dye, 3,3'-diethylthiacarbocyanine iodide ([DiSC.sub.2](3) [I.sup.-]), in the presence of a target genomic DNA and a complementary peptide nucleic acid (PNA) probe. On the basis of the UV-vis, circular dichroism, and fluorescence spectra of [DiSC.sub.2](3) with PNA-DNA oligomer duplexes and on characterization of a product of photolysis of [DiSC.sub.2](3) [I.sup.-], a possible reaction mechanism is proposed. We propose that (1) a novel complex forms between dye, PNA, and DNA, (2) this complex functions as a photosensitizer producing [sup.1][O.sub.2], and (3) the [sup.1][O.sub.2] produced promotes photobleaching of dye molecules in the mixture. Similar cyanine dyes ([DiSC.sub.3](3), [DiSC.sub.4](3), [DiSC.sub.5](3), and [DiSC.sub.py](3)) interact with preformed PNA-DNA oligomer duplexes but do not demonstrate an equivalent accelerated photobleaching effect in the presence of PNA and target genomic DNA. The feasibility of developing molecular diagnostic assays based on the accelerated photobleaching (the smartDNA assay) that results from the novel complex formed between [DiSC.sub.2](3) and PNA-DNA is under way.

A continuous-flow, temperature gradient microfluidic device was used to demonstrate spatial DNA melting analysis with the resolution and reproducibility necessary for clinical SNP scanning and genotyping of human genomic DNA. With a steady-state temperature gradient of 20-30[degrees]C across a sample, melting curves were constructed from a single fluorescence data acquisition. This technique was used to scan for heterozygotes and to fully genotype single base changes using unlabeled probes. Signal-to-noise ratios of 150-300 were achieved. The thermal effects of sample flow were examined, and temperature control was aided by inclusion of an isothermal channel inlet and thermal relaxation times in the experimental protocol. Human single base variants examined by spatial DNA melting analysis included rs354439, HTR2A 102T > C, and three alleles that affect appropriate warfarin dosage ([CYP2C9*2, [CYP2C9*3, and VKORC1 1173C > T). Heterozygote scanning was demonstrated with rs354439, while the other PCR targets were genotyped using unlabeled probes with [T.sub.m] differences of approximately 5[degrees]C between genotypes. To validate the method, 12 blinded DNA samples were genotyped at the three warfarin-related sites by spatial DNA melting analysis with 100% accuracy.

This report highlights methodologies that enable statistically significant data to be collected for single nucleotide polymorphisms using surface enhanced Raman spectroscopy. Single-stranded oligonucleotides functionalized with 40 nm gold nanoparticles are hybridized with oligonucleotides adsorbed to a photolithographically defined gold surface thus creating a surface enhanced Raman environment around the DNA duplex. With this design characteristic Raman spectra have been collected and explored for differences between DNA duplexes formed from complementary oligonucleotides, completely mismatched oligonucleotides, and those formed from oligonucleotides that have a midsequence single nucleotide mismatch. The results show that statistically significant differences in Raman intensity for characteristic peaks can be collected for the three cases.

A class-C floral homeotic gene of Petunia, pMADS3, is specifically expressed in the stamen and carpels of developing flowers. We had previously reported the ect-pMADS3 phenomenon in which introduction of a part of the pMADS3 genomic sequence, including intron 2, induces ectopic expression of endogenous pMADS3. Unlike transcriptional or posttranscriptional gene silencing triggered by the introduction of homologous sequences, this observation is unique in that the gene expression is up-regulated. In this study, we demonstrated that the ect-pMADS3 phenomenon is due to transcriptional activation based on RNA-directed DNA methylation (RdDM) occurring in a particular CG in a putative cis-element in pMADS3 intron 2. The CG methylation was maintained over generations, along with pMADS3 ectopic expression, even in the absence of RNA triggers. These results demonstrate a previously undescribed transcriptional regulatory mechanism that could lead to the generation of a transcriptionally active epiallele, thereby contributing to plant evolution. Our results also reveal a putative negative cis-element for organ-specific transcriptional regulation of class-C floral homeotic genes, which could be difficult to identify by other approaches. epiallele | flower | MADS-box | petunia | RdDM

Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides (dNTPs), the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of dNTPs during the cell cycle. Streptomyces species contain genes, nrdAB and nrdJ, coding for oxygen-dependent class I and oxygen-independent class II RNRs, either of which is sufficient for vegetative growth. Both sets of genes are transcriptionally repressed by NrdR. NrdR contains a zinc ribbon DNA-binding domain and an ATP-cone domain similar to that present in the allosteric activity site of many class I and class III RNRs. Purified NrdR contains up to 1 mol of tightly bound ATP or dATP per mol of protein and binds to tandem 16-bp sequences, termed NrdR-boxes, present in the upstream regulatory regions of bacterial RNR operons. Previously, we showed that the ATP-cone domain alone determines nucleotide binding and that an NrdR mutant defective in nucleotide binding was unable to bind to DNA probes containing NrdR-boxes. These observations led us to propose that when NrdR binds ATP/dATP it undergoes a conformational change that affects DNA binding and hence RNR gene expression. In this study, we analyzed a collection of ATP-cone mutant proteins containing changes in residues inferred to be implicated in nucleotide binding and show that they result in pleiotrophic effects on ATP/dATP binding, on protein oligomerization, and on DNA binding. A model is proposed to integrate these observations.

We present a microfluidic device for multiplexed immunoassays based on DNA-directed immobilization. Because of the versatile building blocks used for this technique, it is possible to build up user configurable protein microarrays within the microfluidic system by means of DNA-directed self-assembly, which can be used for immunoassay applications. We demonstrate the performance of our system by parallel detection of cytokines in a multiplex immunoassay, employing silver deposition labeling and optical read-out.