DNA Sequence Recognition Research Articles

Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases.

Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.

The CRISPR/Cas9 System for Targeted Genome Engineering in Free-Living Fungi: Advances and Opportunities for Lichenized Fungi.

Studies using whole genome sequencing, computational and gene expression, targeted genome engineering techniques for generating site-specific sequence alterations through non-homologous end joining (NHEJ) by genomic double-strand break (DSB) repair pathway with high precision, resulting in gene inactivation have elucidated the complexity of gene expression, and metabolic pathways in fungi. These tools and the data generated are crucial for precise generation of fungal products such as enzymes, secondary metabolites, antibiotics etc. Artificially engineered molecular scissors, zinc finger nucleases (ZFNs), Transcriptional activator-like effector nucleases (TALENs; that use protein motifs for DNA sequence recognition in the genome) and CRISPR associated protein 9 (Cas9;CRISPR/Cas9) system (RNA-DNA recognition) are being used in achieving targeted genome modifications for modifying traits in free-living fungal systems. Here, we discuss the recent research breakthroughs and developments which utilize CRISPR/Cas9 in the metabolic engineering of free-living fungi for the biosynthesis of secondary metabolites, enzyme production, antibiotics and to develop resistance against post-harvest browning of edible mushrooms and fungal pathogenesis. We also discuss the potential and advantages of using targeted genome engineering in lichenized fungal (mycobiont) cultures to enhance their growth and secondary metabolite production in vitro can be complemented by other molecular approaches.

Novel Two-Component System MacRS Is a Pleiotropic Regulator That Controls Multiple Morphogenic Membrane Protein Genes in Streptomyces coelicolor.

As with most annotated two-component systems (TCSs) of Streptomyces coelicolor, the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS, for morphogenesis and actinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using an S. coelicolor strain expressing MacR-Flag fusion protein, identified in vivo targets of MacR, and DNase I footprinting of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNase I footprinting and electrophoretic mobility shift assays in vitro and by ChIP-quantitative PCR in vivo, and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes, sco6728, sco4924, and sco4011, markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein A (MmpA; SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS from S. avermitilis and S. venezuelae, respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism in StreptomycesIMPORTANCE TCSs help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in the Streptomyces model strain S. coelicolor are unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that other Streptomyces species have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems in Streptomyces.

Kinetic and catalytic properties of M.HpyAXVII, a phase-variable DNA methyltransferase from Helicobacter pylori

The bacterium Helicobacter pylori is one of the most common infectious agents found in the human stomach. H. pylori has an unusually large number of DNA methyltransferases (MTases), prompting speculation that they may be involved in the cancerization of epithelial cells. The mod-4a/4b locus, consisting of the hp1369 and hp1370 ORFs, encodes for a truncated and inactive MTase in H. pylori strain 26695. However, slipped-strand synthesis within the phase-variable polyguanine tract in hp1369 results in expression of an active HP1369-1370 fusion N6-adenine methyltransferase, designated M.HpyAXVII. Sequence analysis of the mod-4a/4b locus across 74 H. pylori strain genomes has provided insights into the regulation of M.HpyAXVII expression. To better understand the role of M.HpyAXVII in the H. pylori biology, here we cloned and overexpressed the hp1369-70 fusion construct in Escherichia coli BL21(DE3) cells. Results from size-exclusion chromatography and multi-angle light scattering (MALS) analyses suggested that M.HpyAXVII exists as a dimer in solution. Kinetic studies, including product and substrate inhibition analyses, initial velocity dependence between substrates, and isotope partitioning, suggested that M.HpyAXVII catalyzes DNA methylation in an ordered Bi Bi mechanism in which the AdoMet binding precedes DNA binding and AdoMet's methyl group is then transferred to an adenine within the DNA recognition sequence. Altering the highly conserved catalytic motif (DPP(Y/F)) as well as the AdoMet-binding motif (FXGXG) by site-directed mutagenesis abolished the catalytic activity of M.HpyAXVII. These results provide insights into the enzyme kinetic mechanism of M.HpyAXVII. We propose that AdoMet binding conformationally "primes" the enzyme for DNA binding.

A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing

CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, invivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (N4CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for invivo genome editing applications.

KZF-L162R Mutation Affects Splenic Mature B Cell Development and Alters Expression of Aiolos Target Genes

Large-scale DNA sequencing efforts in chronic lymphocytic leukemia (CLL) have identified a broad array of putative cancer drivers arising from somatic mutations in this disease, but functional understanding of the impact of these genetic events on CLL onset and progression remains to be elucidated. One such example is mutation in the IKZF3 gene, encoding the zinc finger protein AIOLOS, mutated in ~2% of CLLs and associated with fludarabine-refractory disease. AIOLOS is a lymphoid-restricted transcription factor and a chromatin remodeler that plays an essential role in B cell development and maturation. In CLL, the IKZF3 mutation, also reported in few cases of diffuse large B cell lymphoma and mantle cell lymphoma,targets a highly conserved hotspot (L162R, homologous to murine L161R) that is localized in the 2nd zinc finger of the DNA-binding domain, required for DNA sequence recognition. Given the localization of this hotspot mutation, we hypothesized that it impacts the function of AIOLOS to drive CLL.To characterize the effects of the IKZF3-L162R mutation, we generated a knock-in mouse line that conditionally expresses the point mutation in a B cell lineage context through crossing with Cd19-cre mice, generating mouse lines carrying Ikzf3-L161R as either a heterozygous mutation (Ikzf3-L161RHet), homozygous mutation (Ikzf3-L161RHomo) or wild-type Ikzf3(Ikzf3WT).Given the established role of Aiolos in lymphoid differentiation, we first asked how the mutation impacts B cell development. By flow cytometry, using established markers to detect marrow pro-B, pre-B, transitional and mature B cell populations, or peritoneal B1a and B1b cell populations, no differences in the proportion of cells were observed between Ikzf3WT or Ikzf3-L161RHet. In the spleen, however, the average proportion of marginal zone B cells (B220+CD23+CD21high) was markedly reduced in heterozygousmice compared to wild type mice (6 mice/group: 4.9% vs. 11.5%, p=0.006), while the average proportion of follicular B cells (B220+CD23+CD21-) was increased (76% vs. 63%; p=0.003). Immunohistochemical staining of spleen sections confirmed that the marginal zone area was significantly reduced in Ikzf3-L161RHet mice (p=0.01). In addition, we noted a higher proliferation rate of B cells from Ikzf3-L161RHet mice when stimulated with LPS and IL-4 for 3 days (p=0.01), suggesting that the mutation confers a survival advantage to B cells. Similar analyses in Ikzf3-L161RHomo mice are ongoing.By immunofluorescence and immunoprecipitation, neither Aiolos binding with its partners CHD4, SIN3 or HDAC1, nor its cellular distribution were impacted by the mutation. Of note, the total protein level of Aiolos was increased in Ikzf3-L161RHet mice (9 mice/group; p<0.05). Since the mutation localizes to a DNA binding domain, we hypothesized that it modifies the ability of Aiolos to control expression of its target genes. We therefore performed CHIP-seq in Ikzf3WT splenic B cells, and identified Aiolos-associated high confidence peaks (fold change (FC) enrichment compared to input > 20) corresponding to DNA binding sites in the promoters of genes such as Rps19, Ogg1, Dusp2, Phf23 or Brfp1 and confident peaks (FC>10) in the anti-apoptotic gene Mcl1 and in genes involved in BCR signaling (i.e.Syk, Pi3kr1, Nfkbid), suggesting that their expression is under the control of Aiolos.Comparison of the expression by qPCR of these 8 genes in splenic B cells from the 3 mouse lines revealed Dusp2, Mcl1, Syk, Nfkbid and Phf23 to be upregulated in Ikzf3-L161RHomo B cells (p<0.05) but not in Ikzf3-L161RHet B cells. These findings suggest that the mutation directly impacts the expression level of Aiolos target genes. The upregulation of Mcl1 expression is particularly relevant in the context of CLL as dysregulation of anti-apoptotic signaling is characteristic of the disease.In conclusion, these data show that Aiolos mutation affects B cell subpopulation ontogeny, inducing a disproportionate abundance of follicular B cells endowed with high proliferative capacity. The mutation impacts Aiolos transcription capacity leading to upregulation of genes belonging to pathways cardinal to CLL development, including BCR signaling and apoptosis. Ongoing studies focus combining RNA-seq and CHIP-seq in mutant B cells, with the aim of identifying the breadth of differential expressed genes and dysregulated cellular pathways in mutant B cells in an unbiased manner. DisclosuresWu:Neon Therapeutics: Equity Ownership.

Unique mechanism of target recognition by PfoI restriction endonuclease of the CCGG-family

Restriction endonucleases (REs) of the CCGG-family recognize a set of 4–8 bp target sequences that share a common CCGG or CCNGG core and possess PD…D/ExK nuclease fold. REs that interact with 5 bp sequence 5′-CCNGG flip the central N nucleotides and ‘compress’ the bound DNA to stack the inner base pairs to mimic the CCGG sequence. PfoI belongs to the CCGG-family and cleaves the 7 bp sequence 5′-T|CCNGGA ("|" designates cleavage position). We present here crystal structures of PfoI in free and DNA-bound forms that show unique active site arrangement and mechanism of sequence recognition. Structures and mutagenesis indicate that PfoI features a permuted E…ExD…K active site that differs from the consensus motif characteristic to other family members. Although PfoI also flips the central N nucleotides of the target sequence it does not ‘compress’ the bound DNA. Instead, PfoI induces a drastic change in DNA backbone conformation that shortens the distance between scissile phosphates to match that in the unperturbed CCGG sequence. Our data demonstrate the diversity and versatility of structural mechanisms employed by restriction enzymes for recognition of related DNA sequences.

Role of Conformational Fluctuations of Protein toward Methylation in DNA by Cytosine-5-methyltransferase.

Methylation of cytosine is the common epigenetic modification in genomes ranging from bacteria to mammals, and aberrant methylation leads to human diseases including cancer. Recognition of a cognate DNA sequence by DNA methyltransferases and flipping of a target base into the enzyme active site pocket are the key steps in DNA methylation. Using molecular dynamics simulations and enhanced sampling techniques here we elucidate the role of conformational fluctuations of protein and active or passive involvement of protein elements that mediate base flipping and formation of the closed catalytic complex. The free energy profiles for the flipping of target cytosine into the enzyme active site support the major groove base eversion pathway; and the results show that the closed state of enzyme increases the free energy barrier, whereas the open state reduces it. We found that the interactions of the key loop residues of protein with cognate DNA altered the protein motions, and modulation of protein fluctuations relates to the closed catalytic complex formation. Methylation of cytosine in the active site of the closed complex destabilizes the interactions of catalytic loop residues with cognate DNA and reduces the stability of the closed state. Our study provides microscopic insights on the base flipping mechanism coupled with enzyme's loop motions and provides evidence for the role of conformational fluctuations of protein in the enzyme-catalyzed DNA processing mechanism.

Phenotypic Nonspecificity as the Result of Limited Specificity of Transcription Factor Function.

Drosophila transcription factor (TF) function is phenotypically nonspecific. Phenotypic nonspecificity is defined as one phenotype being induced or rescued by multiple TFs. To explain this unexpected result, a hypothetical world of limited specificity is explored where all TFs have unique random distributions along the genome due to low information content of DNA sequence recognition and somewhat promiscuous cooperative interactions with other TFs. Transcription is an emergent property of these two conditions. From this model, explicit predictions are made. First, many more cases of TF nonspecificity are expected when examined. Second, the genetic analysis of regulatory sequences should uncover cis-element bypass and, third, genetic analysis of TF function should generally uncover differential pleiotropy. In addition, limited specificity provides evolutionary opportunity and explains the inefficiency of expression analysis in identifying genes required for biological processes.

Compound Shape Effects in Minor Groove Binding Affinity and Specificity for Mixed Sequence DNA.

AT specific heterocyclic cations that bind in the DNA duplex minor groove have had major successes as cell and nuclear stains and as therapeutic agents which can effectively enter human cells. Expanding the DNA sequence recognition capability of the minor groove compounds could also expand their therapeutic targets and have an impact in many areas, such as modulation of transcription factor biological activity. Success in the design of mixed sequence binding compounds has been achieved with N-methylbenzimidazole ( N-MeBI) thiophenes which are preorganized to fit the shape of the DNA minor groove and H-bond to the -NH of G·C base pairs that project into the minor groove. Initial compounds bind strongly to a single G·C base pair in an AT context with a specificity ratio of 50 ( KD AT-GC/ KD AT) or less and this is somewhat low for biological use. We felt that modifications of compound shape could be used to probe local DNA microstructure in target mixed base pair sequences of DNA and potentially improve the compound binding selectivity. Modifications were made by increasing the size of the benzimidazole N-substituent, for example, by using N-isobutyl instead of N-Me, and by changing the molecular twist by introducing substitutions at specific positions on the aromatic core of the compounds. In both cases, we have been able to achieve a dramatic increase in binding specificity, including no detectible binding to pure AT sequences, without a significant loss in affinity to mixed base pair target sequences.

Novel Strategy for the Investigation on Chirality Selection of Single-Walled Carbon Nanotubes with DNA by Electrochemical Characterization.

There is a correlation between specific bases of DNA molecules and the chirality of single-walled carbon nanotubes (SWNTs), which contributes the recognition ability of DNA toward partner species of chiral SWNTs. A novel strategy of electrochemical characterization is reported here for the investigation on chirality selection of (7,6) and (6,5) SWNTs with various DNA sequences, and it is found that both DNA strand length and sequence composition significantly affected the interaction of chiral SWNTs with DNA. Then (7,6) and (6,5) SWNTs were distinguished from each other with DNA sequences chosen by electrochemical methods, which demonstrated an effective and excellent feasibility for the strategy and presented a new insight into DNA-SWNT applications. This strategy can also be applied to more chiral SWNTs and DNA sequence recognition and may serve as a prescreening method for the recognition and separation of single-chirality SWNTs, which would be a new contribution to the further development of DNA-SWNT hybrids.

Bound Compound, Interfacial Water, and Phenyl Ring Rotation Dynamics of a Compound in the DNA Minor Groove.

DB2277, a heterocyclic diamidine, is a successful design for mixed base pair (bp) DNA sequence recognition. The compound has a central aza-benzimidazole group that forms two H-bonds with a GC bp that has flanking AT bps. The nuclear magnetic resonance structure of the DB2277-DNA complex with an AAGATA recognition site sequence was determined, and here we report extended molecular dynamics (MD) simulations of the structure. DB2277 has two terminal phenyl-amidine groups, one of which is directly linked to the DB2277 heterocyclic core and the other through a flexible -OCH2- group. The flexibly linked phenyl is too far from the minor groove floor to make direct H-bonds but is linked to an AT bp through water-mediated H-bonds. The flexibly linked phenyl-amidine with water-mediated H-bonds to the bases at the floor of the minor groove suggested that it might rotate in time spans accessible in MD. To test this idea, we conducted multimicrosecond MD simulations to determine if these phenyl rotations could be observed for a bound compound. In a 3 μs simulation, highly dynamic torsional motions were observed for the -OCH2-linked phenyl but not for the other phenyl. The dynamics periodically reached a level to allow 180° rotation of the phenyl while it was still bound in the minor groove. This is the first observation of rotation of a phenyl bound to DNA, and the results provide mechanistic details about how a rotation can occur as well as how mixed bp recognition can occur for monomer compounds bound to the minor groove.

Copper distribution networks in Pseudomonas aeruginosa

Bacterial Cu+ homeostasis is based on protein networks that mobilize copper to cellular targets, enabling both metallation of cuproproteins and maintenance of non‐toxic metal levels. Here, we use genome wide expression analysis (RNA‐Seq) to characterize the response of Pseudomonas aeruginosa to external 0.5 mM CuSO4, a condition where cell viability is uncompromised. Analysis of copR and cueR mutant strains revealed that compartmental Cu levels are independently controlled; the cytoplasmic Cu sensor CueR regulates cytoplasmic chaperones and plasma membrane transporters, whereas the two component system CopR/S shows a putative DNA recognition sequence, forming a regulon composed of genes involved in periplasmic Cu‐homeostasis. Response to Cu‐imbalance resulted in distinct transcriptomic landscapes: a pre‐steady‐state (5 min), where general metabolism is slowed down, and a steady‐state (2 h), where general metabolism is restored. Specific Cu homeostasis genes were strongly regulated in both conditions. Our system‐wide analysis revealed induction of three Cu‐efflux systems (a P1B‐ATPase, a porin, and a resistance‐nodulation‐division (RND) system), a putative Cu‐binding periplasmic chaperone and the unusual presence of two cytoplasmic CopZ proteins. Importantly, novel transmembrane transporters probably mediating Cu‐influx were among those largely repressed genes upon Cu‐stress. Three proteins were identified among these: OprC, an outer membrane porin; PA3789, an inner membrane protein; and PA5030, a Met/His rich protein, member of the major facilitator superfamily (MFS) of transporters. Alternatively, genes encoding previously proposed Cu‐importers ccoA and hmtA did not change their expression levels. The occurrence of cell influx transporters is a prerequisite for homeostasis of a given cellular constituent. However, deletion of the putative Cu‐importers OprC and PA1530 does not affect the Cu content of whole cells, suggesting that Pseudomonas has alternative ways to acquire Cu. As Pseudomonas secrete two major siderophores (pyochelin and pyoverdin) to scavenge metals, we hypothesize that those siderophores will constitute an alternative way to import copper. Interestingly, genes involved in both, pyochelin and pyoverdin pathways, are overexpressed when the outer membrane porin is absent, but only pyochelin‐related genes are up‐regulated when the putative inner‐membrane transporters are deleted. This suggests that pyochelin can be the alternative Cu‐import system that Pseudomonas uses under copper‐starving conditions. Summarizing, our studies show a system‐wide network of sensors/regulators, soluble chaperones, and influx/efflux transporters that control the Cu levels in P. aeruginosa compartments.Support or Funding InformationThis work was supported by NIH Grant R01 GM114949 to J.M.A.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch

Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2β2) disorders, sickle cell disease, and β-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to β- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Ourfindings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence.

Pioneer transcription factors specifically target their recognition DNA sequences within nucleosomes. FoxA is the pioneer transcription factor that binds to the ALB1 gene enhancer in liver precursor cells, and is required for liver differentiation in embryos. The ALB1 enhancer DNA sequence is reportedly incorporated into nucleosomes in cells, although the nucleosome structure containing the targeting sites for FoxA has not been clarified yet. In this study, we determined the nucleosome structure containing the ALB1 enhancer (N1) sequence, by cryogenic electron microscopy at 4.0 Å resolution. The nucleosome structure with the ALB1 enhancer DNA is not significantly different from the previously reported nucleosome structure with the Widom 601 DNA. Interestingly, in the nucleosomes, the ALB1 enhancer DNA contains local flexible regions, as compared to the Widom 601 DNA. Consistently, DNaseI treatments revealed that, in the nucleosome, the ALB1 enhancer (N1) DNA is more accessible than the Widom 601 sequence. The histones also associated less strongly with the ALB1 enhancer (N1) DNA than the Widom 601 DNA in the nucleosome. Therefore, the local histone–DNA contacts may be responsible for the enhanced DNA accessibility in the nucleosome with the ALB1 enhancer DNA.

Colorimetric detection of microRNA based on DNAzyme and nuclease-assisted catalytic hairpin assembly signal amplification

Accurate and quantitative analysis of microRNA (miRNA) expression is critical for the diagnostics and theranostics of a disease. Herein, a proof-of-concept of a colorimetric horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) biosensor for miRNA assay based on nuclease-assisted catalytic hairpin assembly (CHA) signal amplification was demonstrated. Duplex-specific nuclease (DSN) was employed to cleave the single-stranded DNA (ssDNA) chimeric probe (CP) on the magnetic bead (MB) surface via hybridization of the CP and target miRNA. The regenerated miRNA can cleave a large number of ssDNA CP to produce CHA initiator sequence fragments. The CP consists of two main regions: a target miRNA recognition DNA sequence at the 5′ end and a CHA initiator (CI) sequence at the 3′ end. The catalyzed assembly process of CHA produces a large amount of G-rich DNA. In the presence of hemin, the G-rich DNA forms G-quadruplex/hemin complex and mimics the horseradish peroxidase activity, which catalyzes a colorimetric reaction. For the proof-of-concept, microRNA-21 (miR-21) was selected as the model target to authenticate this strategy as a versatile assay platform. The proposed strategy allowed quantitation of the sequence specificity of miRNA-21 with a detection limit of 9.2 fM in a dynamic range from 10 fM–1 nM, with an excellent ability to discriminate the differences in miRNAs. Additionally, the miRNA assay in real samples was satisfactory, thereby confirming its applicability. Therefore, this method exhibited a great potential as a miRNA quantification method in biomedical research and clinical diagnosis.

Comparison of Methods of Detection of Exceptional Sequences in Prokaryotic Genomes.

Many proteins need recognition of specific DNA sequences for functioning. The number of recognition sites and their distribution along the DNA might be of biological importance. For example, the number of restriction sites is often reduced in prokaryotic and phage genomes to decrease the probability of DNA cleavage by restriction endonucleases. We call a sequence an exceptional one if its frequency in a genome significantly differs from one predicted by some mathematical model. An exceptional sequence could be either under- or over-represented, depending on its frequency in comparison with the predicted one. Exceptional sequences could be considered biologically meaningful, for example, as targets of DNA-binding proteins or as parts of abundant repetitive elements. Several methods to predict frequency of a short sequence in a genome, based on actual frequencies of certain its subsequences, are used. The most popular are methods based on Markov chain models. But any rigorous comparison of the methods has not previously been performed. We compared three methods for the prediction of short sequence frequencies: the maximum-order Markov chain model-based method, the method that uses geometric mean of extended Markovian estimates, and the method that utilizes frequencies of all subsequences including discontiguous ones. We applied them to restriction sites in complete genomes of 2500 prokaryotic species and demonstrated that the results depend greatly on the method used: lists of 5% of the most under-represented sites differed by up to 50%. The method designed by Burge and coauthors in 1992, which utilizes all subsequences of the sequence, showed a higher precision than the other two methods both on prokaryotic genomes and randomly generated sequences after computational imitation of selective pressure. We propose this method as the first choice for detection of exceptional sequences in prokaryotic genomes.

Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on a polymer chip.

A specific double-stranded DNA sensing system is of great interest for diagnostic and other biomedical applications. Zinc finger domains, which recognize double-stranded DNA, can be engineered to form custom DNA-binding proteins for the recognition of specific DNA sequences. As a proof of concept, a sequence-enabled reassembly of a TEM-1 β-lactamase system (SEER-LAC) was previously demonstrated to develop zinc finger protein (ZFP) arrays for the detection of a double-stranded bacterial DNA sequence. Here, we implemented the SEER-LAC system to demonstrate the direct detection of pathogen-specific DNA sequences present in E. coli O157:H7 on a lab-on-a-chip. ZFPs custom-designed to detect Shiga toxin in E. coli O157:H7 were immobilized on a cyclic olefin copolymer (COC) chip, which can function as a non-PCR based molecular diagnostic device. Pathogen-specific double-stranded DNA was directly detected by using engineered ZFPs immobilized on the COC chip with high specificity, providing a detection limit of 10 fmol of target DNA in a colorimetric assay. Therefore, in this study, we demonstrated the great potential of ZFP arrays on the COC chip for further development of a simple and novel lab-on-a-chip technology for the detection of pathogens.

CLUSTERING ANALYSIS ON THE DESIGN OF COMPLEX DNA NANOSTRUCTURES USING DNA HYBRIDIZATION, SWARM INTELLIGENCE AND GENETIC ALGORITHM

The main objective of this work is to study the clustering techniques with swarm intelligence and genetic algorithm to create new algorithms and to improve the existing algorithms in order to apply them to the issues of design the nanostructure to computationally simulate the DNA sequence recognition process and to achieve an optimised conformation for the complex structure, the relative orientation between motif pattern and DNA hybridisation such that to bind the complementary DNA sequences to form structure. In the design of nanostructure, DNA molecular recognition capacities make DNA an appealing candidate for the construction of novel nanomaterials. The modified Boyer Moore algorithm and modified K-nearest neighbour is for design the complex nanostructures to improve a candidate solution with regard to a given measure of quality.

Structural basis for assembly of the CBF3 kinetochore complex

Eukaryotic chromosomes contain a specialised region known as the centromere, which forms the platform for kinetochore assembly and microtubule attachment. The centromere is distinguished by the presence of nucleosomes containing the histone H3 variant, CENP‐A. In budding yeast, centromere establishment begins with the recognition of a specific DNA sequence by the CBF3 complex. This in turn facilitates CENP‐ACse4 nucleosome deposition and kinetochore assembly. Here, we describe a 3.6 Å single‐particle cryo‐EM reconstruction of the core CBF3 complex, incorporating the sequence‐specific DNA‐binding protein Cep3 together with regulatory subunits Ctf13 and Skp1. This provides the first structural data on Ctf13, defining it as an F‐box protein of the leucine‐rich‐repeat family, and demonstrates how a novel F‐box‐mediated interaction between Ctf13 and Skp1 is responsible for initial assembly of the CBF3 complex.