A single molecule array for digital targeted molecular analyses

We present a new random array format together with a decoding scheme for targeted multiplex digital molecular analyses. DNA samples are analyzed using multiplex sets of padlock or selector probes that create circular DNA molecules upon target recognition. The circularized DNA molecules are amplified through rolling-circle amplification (RCA) to generate amplified single molecules (ASMs). A random array is generated by immobilizing all ASMs on a microscopy glass slide. The ASMs are identified and counted through serial hybridizations of small sets of tag probes, according to a combinatorial decoding scheme. We show that random array format permits at least 10 iterations of hybridization, imaging and dehybridization, a process required for the combinatorial decoding scheme. We further investigated the quantitative dynamic range and precision of the random array format. Finally, as a demonstration, the decoding scheme was applied for multiplex quantitative analysis of genomic loci in samples having verified copy-number variations. Of 31 analyzed loci, all but one were correctly identified and responded according to the known copy-number variations. The decoding strategy is generic in that the target can be any biomolecule which has been encoded into a DNA circle via a molecular probing reaction.     

The mechanism of the search for homology promoted by recA protein. Facilitated diffusion within nucleoprotein networks

recA protein promotes the homologous pairing of single strands with duplex DNA by polymerizing on the single strands to make presynaptic nucleoprotein filaments which are polyvalent with respect to duplex DNA and which consequently form large networks or coaggregates when duplex DNA is added. Previous work has shown that efficient homologous pairing occurs within these networks. In the experiments described here, we observed that the length of the duplex DNA determined the stability of coaggregates, their steady state level, and the yield of joint molecules. Correspondingly, heterologous duplex DNA when preincubated with presynaptic filaments excluded subsequently added homologous duplex DNA from coaggregates and inhibited homologous pairing; the extents of exclusion and inhibition were determined by the length of the heterologous duplex DNA. On the other hand, long heterologous duplex DNA when added together with short homologous duplex DNA was capable of stimulating the absorption of the homologous molecules into coaggregates and increasing the rate of homologous pairing. In reactions involving short duplex molecules, polyamines exerted comparable effects on coaggregation and homologous pairing. We conclude that coaggregates are instrumental in homologous pairing, that they constitute distinct domains that are responsible for the processive or first order character of the pairing reaction, and that they act by concentrating DNA and facilitating diffusion.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.     

Variable copy number of macronuclear DNA molecules in Tetrahymena.

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins.     

CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3

The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is?then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg(2+)-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization.     

The progeny of wingless-expressing cells deliver the signal at a distance in Drosophila embryos

Pattern formation in developing animals requires that cells exchange signals mediated by secreted proteins. How these signals spread is still unclear. It is generally assumed that they reach their target site either by diffusion or active transport (reviewed in [1] [2]). Here, we report an alternative mode of transport for Wingless (Wg), a member of the Wnt family of signaling molecules. In embryos of the fruit fly Drosophila, the wingless (wg) gene is transcribed in narrow stripes of cells abutting the source of Hedgehog protein. We found that these cells or their progeny are free to roam towards the anterior. As they do so, they no longer receive the Hedgehog signal and stop transcribing wg. The cells leaving the expression domain retain inherited Wg protein in secretory vesicles, however, and carry it forwards over a distance of up to four cell diameters. Experiments using a membrane-tethered form of Wg showed that this mechanism is sufficient to account for the normal range of Wg. Nevertheless, evidence exists that Wg can also reach distant target cells independently of protein inheritance, possibly by restricted diffusion. We suggest that both transport mechanisms operate in wild-type embryos.     

Brownian dynamics simulation of knot diffusion along a stretched DNA molecule

Manipulation of individual DNA molecules by optical tweezers has made it possible to tie these molecules into knots. After stretching the DNA molecules the knots become highly localized. In their recent study, Quake and co-authors investigated diffusion of such knots along stretched DNA molecules. We used these data to test the accuracy of a Brownian dynamics simulation of DNA bending motion. We simulated stretched DNA molecules with knots 3(1), 4(1), and 7(1), and determined their diffusion coefficients. Comparison of the simulated and experimental results shows that Brownian dynamics simulation is capable of predicting the rates of large-scale DNA rearrangements within a factor of 2.     

Biological significance of facilitated diffusion in protein-DNA interactions. Applications to T4 endonuclease V-initiated DNA repair

Facilitated diffusion along nontarget DNA is employed by numerous DNA-interactive proteins to locate specific targets. Until now, the biological significance of DNA scanning has remained elusive. T4 endonuclease V is a DNA repair enzyme which scans nontarget DNA and processively incises DNA at the site of pyrimidine dimers which are produced by exposure to ultraviolet (UV) light. In this study we tested the hypothesis that there exists a direct correlation between the degree of processivity of wild type and mutant endonuclease V molecules and the degree of enhanced UV resistance which is conferred to repair-deficient Eshcerichia coli. This was accomplished by first creating a series of endonuclease V mutants whose in vitro catalytic activities were shown to be very similar to that of the wild type enzyme. However, when the mechanisms by which these enzymes search nontarget DNA for its substrate were analyzed in vitro and in vivo, the mutants displayed varying degrees of nontarget DNA scanning ranging from being nearly as processive as wild type to randomly incising dimers within the DNA population. The ability of these altered endonuclease V molecules to enhance UV survival in DNA repair-deficient E. coli then was assessed. The degree of enhanced UV survival was directly correlated with the level of facilitated diffusion. This is the first conclusive evidence directly relating a reduction of in vivo facilitated diffusion with a change in an observed phenotype. These results support the assertion that the mechanisms which DNA-interactive proteins employ in locating their target sites are of biological significance.     

A mathematical model relating diffusion of hydrophobic ions to their adsorption on biological membranes as detected with a microdialyzer

We analyze the diffusion of hydrophobic molecules in a dialysis apparatus with respect to their adsorption on biological membrane vesicles confined to one dialysis chamber. The process is described with a kinetic model, which shows that, depending on the pattern of the adsorption isotherm, the kinetic parameter of the diffusion process through the dialysis membrane is up to two-fold increased by the presence of the adsorbing vesicle surface. The model successfully describes the diffusion of tetraphenylborate and 9-aminoacridine in the presence of chromatophores from photosynthetic membrane, with which they interact with hyperbolic and S-shaped isotherms, respectively.     

Facilitated diffusion of a DNA binding protein on chromatin.

Facilitated diffusion accounts for the rapid rate of association of many bacterial DNA binding proteins with specific DNA sequences in vitro. In this mechanism the proteins bind at random to non-specific sites on the DAN and diffuse (by 'sliding' or 'hopping') along the DNA chain until they arrive at their specific functional sites. We have investigated whether such a mechanism can operate in chromatin by using a bacterial DNA binding protein, Escherichia coli RNA polymerase, that depends on linear diffusion to locate initiation sites on DNA. We have measured the competition between chromatin and its free DNA for the formation of initiation complexes. Only the short linker segments exposed by the removal of histone H1 are available for interaction with the polymerase, but the sparsely distributed promoter sites on the linker DNA of such a polynucleosome chain are located at the same rate as those on DNA. We conclude that the polymerase is free to migrate between the separate linker DNA segments of a polynucleosome chain to reach a promoter site. This chain thus permits the 'hopping' of proteins between neighboring linker segments in their search for a target site on the accessible DNA.     

Uniparental Inheritance of Mitochondrial Genes in Yeast: Dependence on Input Bias of Mitochondrial DNA and Preliminary Investigations of the Mechanism

In Saccharomyces cerevisiae, previous studies on the inheritance of mitochondrial genes controlling antibiotic resistance have shown that some crosses produce a substantial number of uniparental zygotes, which transmit to their diploid progeny mitochondrial alleles from only one parent. In this paper, we show that uniparental zygotes are formed especially when one parent (majority parent) contributes substantially more mitochondrial DNA molecules to the zygote than does the other (minority) parent. Cellular contents of mitochondrial DNA (mtDNA) are increased in these experiments by treatment with cycloheximide, alpha-factor, or the uvsp5 nuclear mutation. In such a biased cross, some zygotes are uniparental for mitochondrial alleles from the majority parent, and the frequency of such zygotes increases with increasing bias. In two- and three-factor crosses the cap1, ery1, and oli1 loci behave coordinately, rather than independently; minority markers tend to be transmitted or lost as a unit, suggesting that the uniparental mechanism acts on entire mtDNA molecules rather than on individual loci. This rules out the possibility that uniparental inheritance can be explained by the conversion of minority markers to the majority alleles during recombination. Exceptions to the coordinate behavior of different loci can be explained by marker rescue via recombination. Uniparental inheritance is largely independent of the position of buds on the zygote. We conclude that it is due to the failure of minority markers to replicate in some zygotes, possibly involving the rapid enzymatic destruction of such markers. We have considered two general classes of mechanisms: (1) random selection of molecules for replication, as for example by competition for replicating sites on a membrane; and (2) differential marking of mtDNA molecules in the two parents, possibly by modification enzymes, followed by a mechanism that "counts" molecules and replicates only the majority type. These classes of models are distinguished genetically by the fact that the first predicts that the output frequency of a given allele among the progeny of a large number of zygotes will approximately equal the average input frequency of that allele, while the second class predicts that any input bias will be amplified in the output. The data suggest that bias amplification does occur. We hypothesize that maternal inheritance of mitochondrial or chloroplast genes in many organisms may depend upon a biased input of organelle DNA molecules, which usually favors the maternal parent, followed by failure of the minority (paternal) molecules to replicate in many or all zygotes.     

Study of the DNA/ethidium bromide interactions on mica surface by atomic force microscope: influence of the surface friction

The influence of mica surface on DNA/ethidium bromide interactions is investigated by atomic force microscopy (AFM). We describe the diffusion mechanism of a DNA molecule on a mica surface by using a simple analytical model. It appears that the DNA diffusion on a mica surface is limited by the surface friction due to the counterion correlations between the divalent counterions condensed on both mica and DNA surfaces. We also study the structural changes of linear DNA adsorbed on mica upon ethidium bromide binding by AFM. It turns out that linear DNA molecules adsorbed on a mica surface are unable to relieve the topological constraint upon ethidium bromide binding. In particular, strongly adsorbed molecules tend to be highly entangled, while loosely bound DNA molecules appear more extended with very few crossovers. Adsorbed DNA molecules cannot move freely on the surface because of the surface friction. Therefore, the topological constraint increases due to the ethidium bromide binding. Moreover, we show that ethidium bromide has a lower affinity for strongly bound molecules due to the topological constraint induced by the surface friction.     

RNA and DNA aptamers in cytomics analysis.

BACKGROUND: The systematic evolution of ligands by exponential enrichment (SELEX) technique is a combinatorial library approach in which DNA or RNA molecules (aptamers) are selected by their ability to bind their protein targets with high affinity and specificity, comparable to that of monoclonal antibodies. In contrast to antibodies conventionally selected in animals, aptamers are generated by an in vitro selection process, and can be directed against almost every target, including antigens like toxins or nonimmunogenic targets, against which conventional antibodies cannot be raised. METHODS: Aptamers are ideal candidates for cytomics, as they can be attached to fluorescent reporters or nanoparticles in order to study biological function by fluorescence microscopy, by flow cytometry, or to quantify the concentration of their target in biological fluids or cells using ELISA, RIA, and Western blot assays. RESULTS: We demonstrate the in vitro selection of anti-kinin B1 receptor aptamers that could be used to determine B1 receptor expression during inflammation processes. These aptamers specifically recognize their target in a Northern-Western blot assay, and bind to their target protein whenever they are exposed in the membrane. CONCLUSIONS: Currently, aptamers are linked to fluorescent reporters. We discuss here the present status and future directions concerning the use of the SELEX technique in cytomics.     

Variable copy number of macronuclear DNA molecules in Tetrahymena

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins. © 1992 Wiley-Liss, Inc.     

Nicking enzyme-based internal labeling of DNA at multiple loci

The labeling of biomolecules has become standard practice in molecular biosciences. Modifications are used for detection, sorting and isolation of small molecules, complexes and entire cells. We have recently reported a method for introducing internal chemical and structural modifications into kbp-sized DNA target substrates that are frequently used in single-molecule experiments. It makes use of nicking enzymes that create single-stranded DNA gaps, which can be subsequently filled with labeled oligonucleotides. Here we provide a detailed protocol and further expand this method. We show that modifications can be introduced at distant loci within one molecule in a simple one-pot reaction. In addition, we achieve labeling on both strands at a specific locus, as demonstrated by F?rster resonance energy transfer (FRET) experiments. The protocol requires an initial cloning of the target substrate (3-5 d), whereas the labeling itself takes 4-6 h. More elaborate purification and verification of label incorporation requires 2 h for each method.