Nucleotide Arrangement Research Articles

An Effective Algorithm Based on Sequence and Property Information for N4-methylcytosine Identification in Multiple Species

Abstract: N4-methylcytosine (4mC) is one of the most important epigenetic modifications, which plays a significant role in biological progress and helps explain biological functions. Although biological experiments can identify potential 4mC sites, they are limited due to the experimental environment and labor-intensive process. Therefore, it is crucial to construct a computational model to identify the 4mC sites. Some computational methods have been proposed to identify the 4mC sites, but some problems should not be ignored, such as those presented as follows: (1) a more accurate algorithm is required to improve the prediction, especially for Matthew’s correlation coefficient (MCC); (2) easier method is needed for clinical research to design medicine or treat disease. Considering these aspects, an effective algorithm using comprehensible encoding in multiple species was proposed in this study. Since nucleotide arrangement and its property information could reflect the sequence structure and function, several feature vectors have been developed based on nucleotide energy information, trinucleotide energy information, and nucleotide chemical property information. Besides, feature effect has been analyzed to select the optimal feature vectors for multiple species. Finally, the optimal feature vectors were inputted into the CatBoost algorithm to construct the identification model. The evaluation results showed that our study obtained the highest MCC, i.e., 2.5%~11.1%, 1.4%~17.8%, 1.1%~7.6%, and 2.3%~18.0% higher than previous models for the A. thaliana, C. elegans, D. melanogaster, and E. coli datasets, respectively. These satisfactory results reflect that the proposed method is available to identify 4mC sites in multiple species, especially for MCC. It could provide a reasonable supplement for biological research.

Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson–Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides—crucial elements for ASO therapeutics under development—have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5′-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Recognition of mixed-sequence double-stranded DNA regions using chimeric Invader/LNA probes.

Development of robust oligonucleotide-based probe technologies, capable of recognizing specific regions of double-stranded DNA (dsDNA) targets, continues to attract considerable attention due to the promise of tools for modulation of gene expression, diagnostic agents, and new modalities against genetic diseases. Our laboratory pursues the development of various strand-invading probes. These include Invader probes, i.e., double-stranded oligonucleotide probes with one or more +1 interstrand zipper arrangements of intercalator-functionalized nucleotides like 2'-O-(pyren-1-yl)methyl-RNA monomers, and chimeric Invader/γPNA probes, i.e., heteroduplex probes between individual Invader strands and complementary γPNA strands. Here we report on the biophysical properties and dsDNA-recognition characteristics of a new class of chimeric probes-chimeric Invader/LNA probes-which are comprised of densely modified Invader strands and fully modified complementary LNA strands. The chimeric Invader/LNA probes form labile and distorted heteroduplexes, due to an apparent incompatibility between intercalating pyrene moieties and LNA strands. In contrast, the individual Invader and LNA strands form very stable duplexes with complementary DNA, which provides the driving force for near-stoichiometric recognition of model double-stranded DNA targets with single base-pair accuracy. The distinctive properties of chimeric Invader/LNA probes unlock exciting possibilities in molecular biology, and diagnostic and therapeutic fields.

Molecular Analysis of Cytochrome Oxidase I in Bemisia tabaci (Gennadius) Populations Collected from Four Districts in the Special Region of Yogyakarta

Bemisia tabaci is an agricultural pest that interferes plant growth, as well as being an insect vector of various types of viruses, one of which is the geminivirus group. B. tabaci is called Cryptic Species Complex due to their similar morphology but has different genetic profiles. Climate change and increased global trading of agricultural products could increase B. tabaci population and lead to the emergence of genetic disparity. This study aims to obtain the latest information on the population homogeneity of B. tabaci in four districts of the Yogyakarta and potential differences on their nucleotide arrangements. Molecular identification was performed using PCR and primers C1-J-2198/L2-N-3914 on B. tabaci. B. tabaci COI gene sequences were then compared using a phylogenetic analysis and similarities of nucleotide bases were determined. Results showed that the populations of B. tabaci in Yogyakarta were still homogeneous showed by biotypes similarity, namely the non-B biotype. B. tabaci from Yogyakarta have nucleotide base similarity of 100% with B. tabaci from Singapore (AY686095) and Thailand (AY686092) and 99.56% with species from Bangladesh (AJ748388). Results also showed no differences in the composition of both nucleotide bases and amino acids from the four districts of B. tabaci sampling location. The homogeneous population of B. tabaci and the high incidence of yellowing disease caused by Begomovirus in chili pepper plants in the Special Region of Yogyakarta prove that it is necessary to review the current methods of controlling B. tabaci pests.

Procedurally Generated Artworks Based on Multiple Sequence Alignment of Orthologous Gene Copies

Abstract In this article, the author presents a novel approach to the procedural generation of artwork series based on multiple sequence alignment of orthologous gene copies. In the strategy developed, nucleotides present in a string of DNA (A, G, C, T) were each assigned to an existing artwork. New visual compositions were then created by collaging columns of pixels from each of the existing four artworks according to the arrangement of nucleotides after orthologous genes were aligned. The resulting outcome was a distinctive set of artworks in which visual differences were governed by nucleotide divergence at genes of interest due to evolutionary processes.

A short overview of the CRISPR-Cas adaptation stage.

CRISPR research began over 30 years ago with the incidental discovery of an unusual nucleotide arrangement in the Escherichia coli genome. It took 20 years to find the main function of CRISPR-Cas systems as an adaptive defence mechanism against invading nucleic acids, and our knowledge of their biology has steadily increased ever since. In parallel, the number of applications derived from CRISPR-Cas systems has risen spectacularly. The CRISPR-based genome editing tool is arguably the most exciting application in both basic and applied research. Lately, CRISPR-Cas research has partially shifted to the least understood aspect of its biology: the ability of CRISPR-Cas systems to acquire new immunities during the so-called adaptation step. To date, the most efficient natural system to readily acquire new spacers is the type II-A system of the gram-positive dairy bacterium Streptococcus thermophilus. The discovery of additional systems able to acquire new spacers will hopefully draw more attention to this step of CRISPR-Cas biology. This review focuses on the breakthroughs that have helped to unravel the adaptation phase and on questions that remain to be answered.

On the Importance of Asymmetry in the Phenotypic Expression of the Genetic Code upon the Molecular Evolution of Proteins

The standard genetic code (SGC) is a mapping between the 64 possible arrangements of the four RNA nucleotides (C, A, U, G) into triplets or codons, where 61 codons are assigned to a specific amino acid and the other three are stop codons for terminating protein synthesis. Aminoacyl-tRNA synthetases (aaRSs) are responsible for implementing the SGC by specifically amino-acylating only its cognate transfer RNA (tRNA), thereby linking an amino acid with its corresponding anticodon triplets. tRNAs molecules bind each codon with its anticodon. To understand the meaning of symmetrical/asymmetrical properties of the SGC, we designed synthetic genetic codes with known symmetries and with the same degeneracy of the SGC. We determined their impact on the substitution rates for each amino acid under a neutral model of protein evolution. We prove that the phenotypic graphs of the SGC for codons and anticodons for all the possible arrangements of nucleotides are asymmetric and the amino acids do not form orbits. In the symmetrical synthetic codes, the amino acids are grouped according to their codonicity, this is the number of triplets encoding a given amino acid. Both the SGC and symmetrical synthetic codes exhibit a probability of occurrence of the amino acids proportional to their degeneracy. Unlike the SGC, the synthetic codes display a constant probability of occurrence of the amino acid according to their codonicity. The asymmetry of the phenotypic graphs of codons and anticodons of the SGC, has important implications on the evolutionary processes of proteins.

The Indigenous endomycorrhizal fungi at salak (Salacca zalacca) plantations in Bali, Indonesia and their colonization of the roots

Abstract. Rai IN, Suada IK, Proborini M, Wiraatmaja IW, Semenov M, Krasnov G. 2019. Indigenous endomycorrhizal fungi at salak (Salacca zalacca) plantations in Bali, Indonesia and their colonization of the roots. Biodiversitas 20: 2410-2416. Cultivation of snake fruit, commonly known as salak usually done organically on dry land with limited fertilizer in Bali. This research aimed to observe and to identify the indigenous endomycorrhizal fungi on salak roots. The exploration was carried out by collecting soil and root samples in salak producing areas in Bali, i.e. Bebandem and Selat of Karangasem Regency, Payangan of Gianyar, and Pupuan of Tabanan Regency. At each location, 9 random samples were taken, resulting in a total of 36 samples. Spore extraction was carried out using a wet filtration technique followed by centrifugation according to the method by Brunndrett et al. (2009). Morphological identification was carried out at the genus and species level using the Manual for Identification of Mycorrhiza Fungi for identifying Vesicular-Arbuscular-Mycorrhiza (VAM) fungi (Schenk and Perez, 1990), while molecular identification was carried out according to Tedersoo et al. (2014). The percentage of root infections was carried out using the coloring method with trypan blue. The results showed there were only two genera of endomycorrhizae (Glomus and Entrophospora) identified at the locations of study sites. The results also showed that samples from Bebandem and Selat regions had 3 Glomus species, Payangan had 3 Glomus species and 1 Entrophospora species, while in Pupuan had only 2 Glomus species. Identification results based on morphological characters showed that all species in the genus Glomus consisted of 3 species, namely Glomus sp-1, Glomus sp-2, and Glomus sp-3, while one species in the genus Entrophospora was Entrophospora sp. Genetic identification results based on the nucleotide arrangement showed that Glomus sp-1 concluded as Glomus cubence, Glomus sp-2 concluded as Glomus custos, and Glomus sp-3 concluded as Glomus indicum, while Entrophospora species concluded as Entrophospora_sp_SH197095.06FU. The average of root colonization/ infection was very high, reaching 93.33% in Bebandem and Selat, 95.00% in Pupuan, and 100% in Payangan. The very high root infection rates indicated that the indigenous endomycorrhiza found in these areas was very adaptive in salak plantation, so there is an opportunity to be developed as biofertilizers.

From fluorescent proteins to fluorogenic RNAs: Tools for imaging cellular macromolecules.

The explosion in genome-wide sequencing has revealed that noncoding RNAs are ubiquitous and highly conserved in biology. New molecular tools are needed for their study in live cells. Fluorescent RNA-small molecule complexes have emerged as powerful counterparts to fluorescent proteins, which are well established, universal tools in the study of proteins in cell biology. No naturally fluorescent RNAs are known; all current fluorescent RNA tags are in vitro evolved or engineered molecules that bind a conditionally fluorescent small molecule and turn on its fluorescence by up to 5000-fold. Structural analyses of several such fluorescence turn-on aptamers show that these compact (30-100 nucleotides) RNAs have diverse molecular architectures that can restrain their photoexcited fluorophores in their maximally fluorescent states, typically by stacking between planar nucleotide arrangements, such as G-quadruplexes, base triples, or base pairs. The diversity of fluorogenic RNAs as well as fluorophores that are cell permeable and bind weakly to endogenous cellular macromolecules has already produced RNA-fluorophore complexes that span the visual spectrum and are useful for tagging and visualizing RNAs in cells. Because the ligand binding sites of fluorogenic RNAs are not constrained by the need to autocatalytically generate fluorophores as are fluorescent proteins, they may offer more flexibility in molecular engineering to generate photophysical properties that are tailored to experimental needs.

Pattern analysis of genetics and genomics: a survey of the state-of-art

The endless enhancement and decreasing charges of a complete human genome have given rise to fast acceptance of genetic and genomic information at both research institutions and clinics. Biologists are enchanting the primary steps in the direction of knowing the locations and functions of all the genes and controlling sites in the genomes of various organisms. As these researchers govern the nucleotide arrangement of large stretches of the human genome, they are constructing excessive volumes of sequence data. Direct research laboratory investigation of this data is expensive and tough, creating computational techniques vital. The arena of pattern analysis, which intends to build computer algorithms that enhance with knowledge, embraces the capacity to empower computers to support humans in the analysis of complex, large genetic and genomic data sets. Here, an overview of pattern analysis techniques for the study of genome sequencing datasets, as well as the proteomics, epigenetic and metabolomic data is delivered. These techniques employ data pre-processing, feature extraction and selection, classification and clustering. The aim of this survey is to present deliberations and recurring challenges in the application of pattern analysis methods, as well as of discriminative and reproductive modeling approaches and discuss the future research directions of these methods for the analysis of genomic and genetic data sets.

GENOMOSYSTEMATICS OF RICKETTSIAE

The definition of the term genome was given by the German botanist G. Winkler almost one hundred years ago in 1920. A genome definition for bacterial (rickettsia) with a single chromosome was recently presented from the perspective of information theory, biology and bioinformatics as the information chain of nucleotides. The systematics of rickettsiae (obligate intracellular microorganisms) is based on a limited number of phenotypic characters. Classifications built on the analysis of genes, fragments of genomes and their concatenations cause discussion. Application of the Formal Order Analysis (FOA, http://foarlab.org) in the study of complete genomes allowed to submit the systematics of representatives of the family Rickettsiaceae. This approach confirmed the existence of typhus group (TG), spotted fever group (SFG), and an «ancestral» group within the genus Rickettsia, and allowed the isolation of the Rickettsia felis group within this genus, located between the «ancestral» group and the SFG and the R. akari group on the border between the SFG group and the genus Orientia. The development of the tools of FOA — «Map of Genes» and «Matrix of Similarity» — helped to conduct an in-depth study of the complete genomes of rickettsia, taking into account the characteristics of their genes and noncoding sequences. Application of these instruments, with the help of the obtained classification, confirmed the notion of ecological features of rickettsia, the structure of nosological forms and the epidemiological patterns of rickettsiosis, and made it possible to assess the virulence of the strains of the two most pathogenic species of rickettsia, R. prowazekii and R. rickettsia. In this work, for the first time, a holistic, consistent and multidimensional observation of a set of closely related bacteria (a family of bacteria) and the manifestations associated with them was carried out. The basis of the developed and herein described systematic approach to the study of bacteria is a new mathematical model — the arrangement of nucleotides in a complete genome and its sensitive unambiguous numerical characteristics. A new methodological approach named genomosystematics and based on mathematical modeling of complete genomes of rickettsiae (bacteria) using FOA. Classification of rickettsiae and rickettsioses obtained with the help of this approach corresponds to ecological, epidemiological and etiological principles. Application of the genomosystematics can serve the goals and objectives of preventive medicine. The publication completes a series of scientific works presenting the methodology of an integrated approach based on the application of mathematical analysis tools in the study of objects and laws of natural science disciplines of biological and medical profile.

New approaches in the systematics of rickettsiae.

The development of a formal order analysis (FOA) allowed constructing a classification of 49 genomes of Rickettsiaceae family representatives. Recently FOA has been extended with new tools—‘Map of genes,’ ‘Matrix of similarity’ and ‘Locality-sensitive hashing’—for a more in-depth study of the structure of rickettsial genomes. The new classification confirmed and supplemented the previously constructed one by determining the position of Rickettsia africae str. ESF-5, R. heilongjiangensis 054, R. monacensis str. IrR/Munich, R. montanensis str. OSU 85-930, R. raoultii str. Khabarovsk, R. rhipicephali str. 3-7-female6-CWPP and Rickettsiales bacterium str. Ac37b. The ‘Map of genes’ demonstrated the complete genomes and their components in a graphical form. The ‘Matrix of similarity’ was applied for an in-depth classification to a subtaxonomic category of the strain within the species R. rickettsii (11 strains) and R. prowazekii (ten strains). The ‘Matrix of similarity’ determines the degree of homology of complete genomes by pairwise comparison of their components and identification of those being identical and similar in the arrangement of nucleotides. A new genomosystematics approach is proposed for the study of complete genomes and their components through the development and application of FOA tools. Its applications include the development of principles for the classification of microorganisms, based on the analysis of complete genomes and their annotations. This approach may help in the taxonomic classification and characterization of some Candidatus Rickettsia spp. that are found in large numbers in arthropods worldwide.

Arrangements of nucleotides flanking the start codon in the IRES of the hepatitis C virus in the IRES binary complex with the human 40S ribosomal subunit

Genomic RNA of hepatitis C virus (HCV) has an internal ribosome entry site (IRES), a specific highly structured fragment responsible for its non-canonical translation initiation. The HCV IRES contains a major part of the 5′-untranslated region of the viral RNA and a small portion of the open reading frame (ORF). At the first step of initiation, IRES directly binds to 40S ribosomal subunits so that the AUG start codon appears at the P site region without scanning and without involving initiation factors. However, it is still not entirely clear whether the IRES ORF is correctly loaded into the 40S ribosomal mRNA binding channel in the resulting binary complex. To address this issue, we applied site-directed cross-linking using HCV IRES derivatives bearing a perfluorophenyl azide cross-linker at nucleotides in definite positions relative to the adenine of the AUG start codon. We found that the modifier at the IRES position −3 cross-links to ribosomal proteins uS11 and eS26. These proteins have been identified together with uS7 as those interacting with the mRNA nucleotide in position −3 relative to the first nucleotide of the codon directed to the P site by a cognate tRNA. Thus, our results indicate a certain difference in the locations of the above parts of HCV IRES and canonical mRNAs on 40S subunits. The modifier at the IRES positions +4/5 was attached to uS19, which is specific for ribosomal complexes with the P site tRNA and similar derivatives of model canonical mRNAs when the modifier is in the same positions. However, the cross-linking efficiency of the IRES derivative was drastically lower than that previously observed with derivatives of model mRNAs. This implies that the IRES ORF portion is correctly loaded into the mRNA binding channel only in a tiny fraction of the binary complexes.

Phlebotomus (Adlerius) kabulensis (Diptera: Psychodidae) a new record sand fly species from Iran: Morphological and molecular aspects

Objective: To represent a new geographical record, Phlebotomus (Adlerius) kabulensis (P. kabulensis), which is suspected to be a potential vector of visceral leishmaniasis. Methods: For the first time, P. kabulensis specimens were collected using the sticky paper traps method in outdoor places in mountainous areas with vegetation coverage of three provinces in Iran. Identification of males was based on ecological, morphological, morphometric and molecular (mtDNA cytochrome b gene sequences) criteria. Generally, males have two ascoids on the 8th antennal segment and one ascoid on segments 9th to 15th, aedeagus with normal obtuse-angled sub-terminal notch and coxite with 27–50 groups of hairs on the distal half of its body. Results: Morphometric measurement revealed that P. kabulensis specimens were the same as compared with seven other morphological characters in three provinces of the country but lengths of the coxite were significantly different. The PCR result of the cytochrome b (Cyt b)-mtDNA fragment shows 550-bp length, with its special nucleotide arrangement. The male and female of P. kabulensis were newly discovered members of the subgenus Adlerius from Iran. Initial DNA analysis indicated how distinct this species is. Conclusions: The results show that the P. kabulensis female will be identified by comparing with other Adlerius female groups regarding its morphometric characters and molecular sequencing.

Structural and Biochemical Properties of Novel Self-Cleaving Ribozymes.

Fourteen well-defined ribozyme classes have been identified to date, among which nine are site-specific self-cleaving ribozymes. Very recently, small self-cleaving ribozymes have attracted renewed interest in their structure, biochemistry, and biological function since the discovery, during the last three years, of four novel ribozymes, termed twister, twister sister, pistol, and hatchet. In this review, we mainly address the structure, biochemistry, and catalytic mechanism of the novel ribozymes. They are characterized by distinct active site architectures and divergent, but similar, biochemical properties. The cleavage activities of the ribozymes are highly dependent upon divalent cations, pH, and base-specific mutations, which can cause changes in the nucleotide arrangement and/or electrostatic potential around the cleavage site. It is most likely that a guanine and adenine in close proximity of the cleavage site are involved in general acid-base catalysis. In addition, metal ions appear to play a structural rather than catalytic role although some of their crystal structures have shown a direct metal ion coordination to a non-bridging phosphate oxygen at the cleavage site. Collectively, the structural and biochemical data of the four newest ribozymes could contribute to advance our mechanistic understanding of how self-cleaving ribozymes accomplish their efficient site-specific RNA cleavages.

Identification of long non-coding transcripts with feature selection: a comparative study

BackgroundThe unveiling of long non-coding RNAs as important gene regulators in many biological contexts has increased the demand for efficient and robust computational methods to identify novel long non-coding RNAs from transcripts assembled with high throughput RNA-seq data. Several classes of sequence-based features have been proposed to distinguish between coding and non-coding transcripts. Among them, open reading frame, conservation scores, nucleotide arrangements, and RNA secondary structure have been used with success in literature to recognize intergenic long non-coding RNAs, a particular subclass of non-coding RNAs.ResultsIn this paper we perform a systematic assessment of a wide collection of features extracted from sequence data. We use most of the features proposed in the literature, and we include, as a novel set of features, the occurrence of repeats contained in transposable elements. The aim is to detect signatures (groups of features) able to distinguish long non-coding transcripts from other classes, both protein-coding and non-coding. We evaluate different feature selection algorithms, test for signature stability, and evaluate the prediction ability of a signature with a machine learning algorithm. The study reveals different signatures in human, mouse, and zebrafish, highlighting that some features are shared among species, while others tend to be species-specific. Compared to coding potential tools and similar supervised approaches, including novel signatures, such as those identified here, in a machine learning algorithm improves the prediction performance, in terms of area under precision and recall curve, by 1 to 24%, depending on the species and on the signature.ConclusionsUnderstanding which features are best suited for the prediction of long non-coding RNAs allows for the development of more effective automatic annotation pipelines especially relevant for poorly annotated genomes, such as zebrafish. We provide a web tool that recognizes novel long non-coding RNAs with the obtained signatures from fasta and gtf formats. The tool is available at the following url: http://www.bioinformatics-sannio.org/software/.

Superstatistical model of bacterial DNA architecture

Understanding the physical principles that govern the complex DNA structural organization as well as its mechanical and thermodynamical properties is essential for the advancement in both life sciences and genetic engineering. Recently we have discovered that the complex DNA organization is explicitly reflected in the arrangement of nucleotides depicted by the universal power law tailed internucleotide interval distribution that is valid for complete genomes of various prokaryotic and eukaryotic organisms. Here we suggest a superstatistical model that represents a long DNA molecule by a series of consecutive ~150 bp DNA segments with the alternation of the local nucleotide composition between segments exhibiting long-range correlations. We show that the superstatistical model and the corresponding DNA generation algorithm explicitly reproduce the laws governing the empirical nucleotide arrangement properties of the DNA sequences for various global GC contents and optimal living temperatures. Finally, we discuss the relevance of our model in terms of the DNA mechanical properties. As an outlook, we focus on finding the DNA sequences that encode a given protein while simultaneously reproducing the nucleotide arrangement laws observed from empirical genomes, that may be of interest in the optimization of genetic engineering of long DNA molecules.

Применение числовой характеристики строя нуклеотидов в геномах прокариот для реклассификации внутри рода Rickettsia

Genomes representing Rickettsiaceae family were analyzed using formal order analysis (FOA) of information chain in order to develop a new approach for the classification of prokaryotes. Average remoteness – the numerical characteristic of order was used to compare the genomes. FOA allows one to directly take into account arrangement of nucleotides in each sequence. The obtained results clarified the previously known classification. In addition Rickettsia felis group was discovered between the ancestral group and spotted fever group (SFG) and R. akari group located between the SFG and genus Orientia. Software used for the analysis of nucleotide sequences with FOA is freely available at http://foarlab.org.

Merging Two Strategies for Mixed-Sequence Recognition of Double-Stranded DNA: Pseudocomplementary Invader Probes.

The development of molecular strategies that enable recognition of specific double-stranded DNA (dsDNA) regions has been a longstanding goal as evidenced by the emergence of triplex-forming oligonucleotides, peptide nucleic acids (PNAs), minor groove binding polyamides, and—more recently—engineered proteins such as CRISPR/Cas9. Despite this progress, an unmet need remains for simple hybridization-based probes that recognize specific mixed-sequence dsDNA regions under physiological conditions. Herein, we introduce pseudocomplementary Invader probes as a step in this direction. These double-stranded probes are chimeras between pseudocomplementary DNA (pcDNA) and Invader probes, which are activated for mixed-sequence dsDNA-recognition through the introduction of pseudocomplementary base pairs comprised of 2-thiothymine and 2,6-diaminopurine, and +1 interstrand zipper arrangements of intercalator-functionalized nucleotides, respectively. We demonstrate that certain pseudocomplementary Invader probe designs result in very efficient and specific recognition of model dsDNA targets in buffers of high ionic strength. These chimeric probes, therefore, present themselves as a promising strategy for mixed-sequence recognition of dsDNA targets for applications in molecular biology and nucleic acid diagnostics.

Conserved sections of the transcription regulatory modules in Drosophila early genes, including homotypic transcription factor-binding sites, are arranged with an 84-nt period, which corresponds to the superhelical turn length of nucleosomal DNA

The periodicity of nucleotide arrangement was studied for conserved regions of the regulatory modules of 16 early development genes in two Drosophila species. The relative disposition of the conserved segments was found to be a multiple of the length of one coil of the nucleosomal DNA superhelix. Homotypic and heterotypic clusters of transcription-factor binding sites were observed in the conserved regions. The sites are usually at a distance that corresponds to the coil length of superhelical DNA of the nucleosome. The transcription-factor binding sites were assumed to play a role in regulating DNA condensation according to the nucleosome type.