DNA Sequence Selectivity Research Articles

Chemical Approaches to the Development of Artificial Transcription Factors Based on Pyrrole‐Imidazole Polyamides

To maintain the functions of living organisms, cells have developed complex gene regulatory networks. Transcription factors have a central role in spatiotemporal control of gene expression and this has motivated us to develop artificial transcription factors that mimic their function. We found that three functions could be mimicked by applying our chemical approaches: i) efficient delivery into organelles that contain target DNA, ii) specific DNA binding to the target genomic region, and iii) regulation of gene expression by interaction with other transcription coregulators. We chose pyrrole-imidazole polyamides (PIPs), sequence-selective DNA binding molecules, as DNA binding domains, and have achieved each of the required functions by introducing other functional moieties. The developed artificial transcription factors have potential as chemical tools that can be used to artificially modulate gene expression to enable cell fate control and to correct abnormal gene regulation for therapeutic purposes.

BiasAway: command-line and web server to generate nucleotide composition-matched DNA background sequences.

MotivationAccurate motif enrichment analyses depend on the choice of background DNA sequences used, which should ideally match the sequence composition of the foreground sequences. It is important to avoid false positive enrichment due to sequence biases in the genome, such as GC-bias. Therefore, relying on an appropriate set of background sequences is crucial for enrichment analysis.ResultsWe developed BiasAway, a command line tool and its dedicated easy-to-use web server to generate synthetic sequences matching any k-mer nucleotide composition or select genomic DNA sequences matching the mononucleotide composition of the foreground sequences through four different models. For genomic sequences, we provide precomputed partitions of genomes from nine species with five different bin sizes to generate appropriate genomic background sequences.Availability and implementationBiasAway source code is freely available from Bitbucket (https://bitbucket.org/CBGR/biasaway) and can be easily installed using bioconda or pip. The web server is available at https://biasaway.uio.no and a detailed documentation is available at https://biasaway.readthedocs.io.Supplementary informationSupplementary data are available at Bioinformatics online.

An integrative understanding of comparative cognition: lessons from human brain evolution.

A comprehensive understanding of animal cognition requires the integration of studies on behavior, electrophysiology, neuroanatomy, development, and genomics. Although studies of comparative cognition are receiving increasing attention from organismal biologists, most current studies focus on the comparison of behaviors and anatomical structures to understand their adaptative values. However, to understand the most potentially complex cognitive program of the human brain a greater synthesis of a multitude of disciplines is needed. In this review, we start with extensive neuroanatomic comparisons between humans and other primates. One likely specialization of the human brain is the expansion of neocortex, especially in regions for high-order cognition (e.g., prefrontal cortex). We then discuss how such an expansion can be linked to heterochrony of the brain developmental program, resulting in a greater number of neurons and enhanced computational capacity. Furthermore, alteration of gene expression in the human brain has been associated with positive selection in DNA sequences of gene regulatory regions. These results not only imply that genes associated with brain development are a major factor in the evolution of cognition, but also that high-quality whole-genome sequencing and gene manipulation techniques are needed for an integrative and functional understanding of comparative cognition in non-model organisms.

Blooms of Toxic Cyanobacterium Nodularia spumigena in Norwegian Fjords During Holocene Warm Periods.

In paleoecological studies, molecular markers are being used increasingly often to reconstruct community structures, environmental conditions and ecosystem changes. In this work, nodularin, anabaenopeptins and selected DNA sequences were applied as Nodularia spumigena markers to reconstruct the history of the cyanobacterium in the Norwegian fjords. For the purpose of this study, three sediment cores collected in Oslofjorden, Trondheimsfjorden and Balsfjorden were analyzed. The lack of nodularin in most recent sediments is consistent with the fact that only one report on the sporadic occurrence and low amounts of the cyanobacterium in Norwegian Fjords in 1976 has been published. However, analyses of species-specific chemical markers in deep sediments showed that thousands of years ago, N. spumigena constituted an important component of the phytoplankton community. The content of the markers in the cores indicated that the biomass of the cyanobacterium increased during the warmer Holocene periods. The analyses of genetic markers were less conclusive; they showed the occurrence of microcystin/nodularin producing cyanobacteria of Nostocales order, but they did not allow for the identification of the organisms at a species level.

Unveiling the Antimicrobial Activities of Dicationic Carbazoles and Related Analogs Through Computational Docking

Computational docking is a globally used tool now-a-days in bioinformatics. All the drugs/ligands generate their effect only when they interact/bind with the target molecule, here DNA. The potential drugs/ligands can only be identified by the study of their relative binding energies and preferential binding modes. Due to availability of huge numbers of such drugs/ligands; the evaluation of their relative potency is a challenging task. In the present work, carbazoles and its derivatives were studied for their DNA binding abilities using computational molecular docking. All the docked ligands had planar structures which allowed them to adopt crescent shape and thus minor groove binding to DNA was preferred by most of them. Computational docking revealed that DNA binding energies of carbazoles and its analogs followed the same trend as their thermal melting values. Also the drugs/ligands preferred themselves to bind at AT-rich regions of the minor groove of the selected DNA sequences.

Modification of polyacrylate sorbent coatings with carbodiimide crosslinker chemistry for sequence-selective DNA extraction using solid-phase microextraction.

Selective DNA extraction is immensely useful for the isolation and detection of low-abundance sequences. Oligonucleotide-modified substrates are often used to capture sequences of interest for downstream analysis. In this study, we explore the chemical modification of commercial-available polyacrylate solid-phase microextraction fibers for selective DNA analysis using carbodiimide crosslinker chemistry. Reproducible modification conditions are found and the fibers were subsequently applied for selective DNA analysis. Several experimental parameters such as stir-rate, desorption time, and buffer-type are optimized. The developed method was able to selectively extract the target DNA sequence (260 bp) in the presence of 100-fold excess interfering salmon testes DNA.

Genome reading by the NF-κB transcription factors.

The NF-κB family of dimeric transcription factors regulates transcription by selectively binding to DNA response elements present within promoters or enhancers of target genes. The DNA response elements, collectively known as κB sites or κB DNA, share the consensus 5'-GGGRNNNYCC-3' (where R, Y and N are purine, pyrimidine and any nucleotide base, respectively). In addition, several DNA sequences that deviate significantly from the consensus have been shown to accommodate binding by NF-κB dimers. X-ray crystal structures of NF-κB in complex with diverse κB DNA have helped elucidate the chemical principles that underlie target selection in vitro. However, NF-κB dimers encounter additional impediments to selective DNA binding in vivo. Work carried out during the past decades has identified some of the barriers to sequence selective DNA target binding within the context of chromatin and suggests possible mechanisms by which NF-κB might overcome these obstacles. In this review, we first highlight structural features of NF-κB:DNA complexes and how distinctive features of NF-κB proteins and DNA sequences contribute to specific complex formation. We then discuss how native NF-κB dimers identify DNA binding targets in the nucleus with support from additional factors and how post-translational modifications enable NF-κB to selectively bind κB sites in vivo.

An integrated pathway for building regional phylogenies for ecological studies

AbstractAimPhylogenies are increasingly used in community ecology, biogeography and macroecology. However, sourcing a phylogeny comprising the entire species pool for a focal region can be difficult. Typically, a bespoke phylogeny must be created requiring considerable data manipulation and the use of many standalone software packages. Here we present a suite of methodological tools within the popular R environment that help to build molecular phylogenies appropriate for ecological studies with a regional focus.InnovationOur R package regPhylo provides a pipeline to construct a Bayesian posterior distribution of time‐calibrated trees suitable to address ecological questions. The novel contributions of regPhylo include options to: use prior phylogenetic knowledge through flexible topological constraints; include spatial metadata in sourcing DNA sequences; and include taxa without DNA sequences and then infer consequent phylogenetic uncertainty. Specifically, regPhylo helps researchers: retrieve DNA sequences; enhance available metadata; select DNA sequences based on their length or spatial proximity to the region of study; align sequences; and perform quality control. Output from the pipeline is a file ready to run in the Bayesian tree reconstruction software beast2, appropriate for estimating time‐calibrated trees and including phylogenetic uncertainty for downstream analyses.Main conclusionsOverall, regPhylo improves the integration of popular standalone phylogenetic software into the flexible R environment. It provides a novel approach to include topological constraints based on prior knowledge, include taxa without DNA sequences, and select spatially appropriate DNA sequences. When coupled with a Bayesian tree‐building process, our approach provides estimates of uncertainty in both topology and branch lengths. We demonstrate the utility of the package by constructing a posterior distribution of time‐calibrated phylogenies for the New Zealand marine ray‐finned fishes (Actinopterygii) providing the unprecedented opportunity to include phylogenetic information in downstream ecological analyses for marine fishes in this region.

Targeting an Artificial Metal Nuclease to DNA by a Simple Chemical Modification and Its Drastic Effect on Catalysis.

A novel metal complex was synthesized containing a purine derived ligand in order to increase its binding to DNA. We observed a huge increase in nuclease activity and, quite interestingly, an improvement on DNA sequence selectivity. A potential site of specific cleavage in the presence of a reductant in the reaction medium is suggested. We were able to synthesize a novel metal nuclease with improved activity on DNA, and with sequence specificity when exposed to a coreactant, this opens up new possibilities to create site specific and redox status modulated artificial nucleases.

Abstract 211: A new class of sequence-selective DNA cross-linking ADC payloads with increased in vivotolerability

Abstract Although five Antibody-Drug Conjugates (ADCs) have been approved and over eighty others are in development, the majority contain payloads belonging to two classes: tubulin inhibitors and DNA interactive agents. Most DNA cross-linking payloads (e.g., the PBD dimers) have potent cytotoxicity but ADCs containing them have high hydrophobicity and a narrow therapeutic window. Thus, there is interest in developing novel payloads which benefit from a potency similar to the PBD dimers but that possess lower hydrophobicity and produce ADCs with a wider Therapeutic Index (TI). The pyridinobenzodiazepines (PDDs) are a new class of guanine-alkylating payloads, and these have been coupled to an adenine-alkylating CXI/duocarmycin pharmacophore to generate molecules that can form G-A DNA cross-links. The lead PDD-CXI payload (FGX-8-46) has a sequence-selectivity profile that differs from other DNA cross-linking agents in that it spans seven to eight base-pairs compared to six to seven for a typical PBD dimer. DNA cleavage experiments have indicated that it cleaves at discrete Adenine-containing sequences of the type 5’-XGXWWWW-3’ (X is any base; W is A/T, and the underlined bases show the cleavage points), and Transcription Factor (TF) Array studies show that it is a potent TF inhibitor, down-regulating several key oncogenic TFs (e.g., NF-κB). In in vitro cell line studies, the G-A cross-linkers have low pM cytotoxicity comparable to the PBD dimers in a wide and diverse range of cell lines, including those from both solid and haematological cancers (e.g., IC50 of ~2 pM in SW-48). This payload class is also compatible with a wide variety of linker technologies, and attachment can be made through either the PDD or CXI units. Importantly, these payloads are significantly less hydrophobic than other equivalent payload classes. ADCs have been generated by conjugating these new payloads to the EGFR-targeting antibody Cetuximab with DARs of between 1.8 and 2.2. The ADCs exhibit potent cytotoxicity in vitro, significant in vivo efficacy and substantially increased tolerability compared to other DNA cross-linking payloads (e.g., the PBD dimer Tesirine). While the G-A cross-linkers retain the cytotoxic potency of the PBD dimers, the “softer” cross-link formed compared to the G-G cross-linking PBD dimers may contribute to the enhanced tolerability profile of this molecular class. The favourable hydrophobicity profile of the PDD-CXI payloads and their ease of conjugation to antibodies, along with their significant in vitro cytotoxicity, in vivo efficacy and tolerability of ADCs produced from them, suggest that they represent a promising new class of ADC payloads. Citation Format: George Procopiou, Jennifer Auer, Daniella di Mascio, Keith R. Fox, Paolo Andriollo, Ilona Pysz, Francesco Cascio, Nicolas Veillard, K. Miraz Rahman, Paul J. Jackson, David E. Thurston. A new class of sequence-selective DNA cross-linking ADC payloads with increased in vivotolerability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 211.

Heterocyclic Diamidine DNA Ligands as HOXA9 Transcription Factor Inhibitors: Design, Molecular Evaluation, and Cellular Consequences in a HOXA9-Dependant Leukemia Cell Model.

Most transcription factors were for a long time considered as undruggable targets because of the absence of binding pockets for direct targeting. HOXA9, implicated in acute myeloid leukemia, is one of them. To date, only indirect targeting of HOXA9 expression or multitarget HOX/PBX protein/protein interaction inhibitors has been developed. As an attractive alternative by inhibiting the DNA binding, we selected a series of heterocyclic diamidines as efficient competitors for the HOXA9/DNA interaction through binding as minor groove DNA ligands on the HOXA9 cognate sequence. Selected DB818 and DB1055 compounds altered HOXA9-mediated transcription in luciferase assays, cell survival, and cell cycle, but increased cell death and granulocyte/monocyte differentiation, two main HOXA9 functions also highlighted using transcriptomic analysis of DB818-treated murine Hoxa9-transformed hematopoietic cells. Altogether, these data demonstrate for the first time the propensity of sequence-selective DNA ligands to inhibit HOXA9/DNA binding both in vitro and in a murine Hoxa9-dependent leukemic cell model.

Rapid Quantitative Evaluation of CRISPR Genome Editing by TIDE and TIDER.

Current genome editing tools enable targeted mutagenesis of selected DNA sequences in many species. However, the efficiency and the type of introduced mutations by the genome editing method are largely dependent on the target site. As a consequence, the outcome of the editing operation is difficult to predict. Therefore, a quick assay to quantify the frequency of mutations is vital for a proper assessment of genome editing actions. We developed two methods that are rapid, cost-effective, and readily applicable: (1) TIDE, which can accurately identify and quantify insertions and deletions (indels) that arise after introduction of double strand breaks (DSBs); (2) TIDER, which is suited for template-mediated editing events including point mutations. Both methods only require a set of PCR reactions and standard Sanger sequencing runs. The sequence traces are analyzed by the TIDE or TIDER algorithm (available at https://tide.nki.nl or https://deskgen.com ). The routine is easy, fast, and provides much more detailed information than current enzyme-based assays. TIDE and TIDER accelerate testing and designing of DSB-based genome editing strategies.

Impedimetric Nanobiosensor for the Detection of Sequence-Selective DNA Hybridization

5-amino-2-mercapto-1,3,4-thiadiazole (AMT) enriched gold nanoparticles (AuNPs) modified impedimetric sensors were developed for the electrochemical monitoring of sequence-selective DNA hybridization related to Hepatitis B virus (HBV). AMT-AuNP-PGEs presented more repeatable impedimetric responses and provided a suitable surface for more DNA binding onto AMT-AuNPs-PGE surface. The selectivity of DNA biosensor modified with AMT-AuNP was investigated in the presence of target DNA, or the other DNA sequences; e.g, noncomplementary (NC), or mismatch (MM) DNA sequences. The detection limit was calculated as 0.86 µg/mL.

ALS genosensing using DNA-hybridization electrochemical biosensor based on label-free immobilization of ssDNA on Sm2O3 NPs-rGO/PANI composite

A DNA-hybridization electrochemical biosensor (DNA-HEB) has been developed using a new platform for immobilization of a 20-mer oligonucleotide. In a DNA-HEB, the signal is produced based on hybridization of a probe and its target sequence. DNA-HEB can be a cost-effective techniques be able to be miniaturized. The selected DNA sequence is related to a gene sequence encoding the Cu-Zn superoxide dismutase enzyme (SOD1) which is important in familial Amyotrophic lateral sclerosis (ALS) disease. Tris(bipyridine)ruthenium(II) chloride was utilized as an electroactive probe to detect the interactions of ssDNA and dsDNA on the electrode surface indirectly. Modifying a glassy carbon electrode with polyaniline and samarium oxide nanoparticles decorated reduced graphene oxide composite, provided a matrix for a lable-free immobilization of ssDNA and enhanced the sensor signal obtained from Fast Fourier Transform square wave voltammetry (FFT-SWV). The studies showed a good dynamic response range from 1 × 10−13 to 1 × 10−8 mol L-1 for the proposed sequence SOD1.

Abstract 736: Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity

Abstract Although four ADCs have been approved and over sixty others are in development, the majority contain payloads belonging to two classes; tubulin inhibitors and DNA cross-linkers. Challenges in the development of ADCs include managing off-target toxicity and hydrophobicity. Some payload classes (e.g., PBD dimers) are notably hydrophobic leading to problems (e.g., aggregation) during conjugation. Thus, there is interest in developing novel payloads which retain the potency of DNA cross-linkers but have lower hydrophobicity and a wider therapeutic window when part of an ADC. The pyridinobenzodiazepines (PDDs) are a new class of sequence-selective, DNA mono-alkylating ADC payload which contain a polyheterocyclic chain with sufficient span to guide them to specific DNA sequences (e.g., transcription factor binding sites). The lead PDD payload, FGX-2-62, has a different sequence-selectivity profile to other DNA-interactive agents, spanning 8-9 base-pairs compared to 6-7 for a PBD dimer, and DNA footprinting experiments indicate a preference for 5'-XGXWWWWXX-3' sequences (X is any base; W is A/T). Transcription factor array studies have shown that the molecule inhibits DNA-binding of oncogenic transcription factors (e.g., NF-κB and GATA). In in vitro cell line studies, FGX-2-62 has low pM cytotoxicity in a diverse cell line panel, including stem cells, cells from both solid and blood cancers (e.g., 9 pM in HL-60) and MDR-resistant tumours, and arrests the cell cycle at the G0/G1 phase compared to G2-M arrest for PBD dimers. It is compatible with attachment to most linker technologies, and is significantly less hydrophobic than other payload classes. Initial MTD studies were carried out by separately conjugating (with negligible aggregation) FGX-2-62 and the PBD dimer Talirine to a THIOMAB® version of trastuzumab (DAR = 2). In female athymic nude mice, a greater tolerance was observed for the THIOMAB®-(FGX-2-62) ADC compared to the THIOMAB®-PBD dimer (i.e, MTD >8 mg.kg-1 versus 4 mg.kg-1). In an efficacy study, FGX-2-62 was conjugated to a cancer stem cell-targeting IgG1 antibody (Bstrongximab) with DAR 1.9. Initial evaluation afforded IC50 values of 0.67 nM and 0.47 nM in two antigen positive cell-lines, and an MTD of 6 mg.kg-1 in mice. In an antigen-positive embryonal carcinoma stem cell CDX mouse model, complete regression was observed at a dose of 2 mg.kg-1 (Q7Dx3). In a cholangiocarcinoma PDX model, complete tumour regression was observed out to 80 days (when experiment was terminated) at a dose of 5 mg.kg-1 (Q7Dx3), with no observed toxicity. The favourable hydrophobicity profile of the PDDs and ease of conjugation, along with their novel mechanism of action, significant in vitro cytotoxicity, in vivo efficacy and tolerability in MTD studies suggest that they represent a promising new class of ADC payloads. Citation Format: Nicolas Veillard, Paolo Andriollo, Julia Mantaj, Keith R. Fox, K Miraz Rahman, George Procopiou, Francesco Cascio, David B. Corcoran, Ilona Pysz, Patricia A. Cooper, Steven D. Shnyder, Yawen Ju, Edwin Tan, William M. Schopperle, Paul J. Jackson, David E. Thurston. Pyridinobenzodiazepines (PDDs): A new class of sequence-selective DNA mono-alkylating ADC payloads with low hydrophobicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 736.

Structure-Defined DNA-Carbon Nanotube Hybrids and Their Applications

In this paper, I present first our current understanding of structure-defined DNA-carbon nanotube hybrids, then a description of the DNA sequence selection problem, and finally utility of DNA-carbon nanotube hybrids in molecular sensing. An artificial perception system, i.e. molecular perceptron, is proposed to take full advantage of the structure diversity of DNA-carbon nanotube hybrids.

Biomimetic Artificial Epigenetic Code for Targeted Acetylation of Histones.

While the central role of locus-specific acetylation of histone proteins in eukaryotic gene expression is well established, the availability of designer tools to regulate acetylation at particular nucleosome sites remains limited. Here, we develop a unique strategy to introduce acetylation by constructing a bifunctional molecule designated Bi-PIP. Bi-PIP has a P300/CBP-selective bromodomain inhibitor (Bi) as a P300/CBP recruiter and a pyrrole-imidazole polyamide (PIP) as a sequence-selective DNA binder. Biochemical assays verified that Bi-PIPs recruit P300 to the nucleosomes having their target DNA sequences and extensively accelerate acetylation. Bi-PIPs also activated transcription of genes that have corresponding cognate DNA sequences inside living cells. Our results demonstrate that Bi-PIPs could act as a synthetic programmable histone code of acetylation, which emulates the bromodomain-mediated natural propagation system of histone acetylation to activate gene expression in a sequence-selective manner.

Computational Prediction of Sigma-54 Promoters in Bacterial Genomes by Integrating Motif Finding and Machine Learning Strategies.

Sigma factor, as a unit of RNA polymerase holoenzyme, is a critical factor in the process of gene transcriptional regulation. It recognizes the specific DNA sites and brings the core enzyme of RNA polymerase to the upstream regions of target genes. Therefore, the prediction of the promoters for a particular sigma factor is essential for interpreting functional genomic data and observation. This paper develops a new method to predict sigma-54 promoters in bacterial genomes. The new method organically integrates motif finding and machine learning strategies to capture the intrinsic features of sigma-54 promoters. The experiments on E. coli benchmark test set show that our method has good capability to distinguish sigma-54 promoters from surrounding or randomly selected DNA sequences. The applications of the other three bacterial genomes indicate the potential robustness and applicative power of our method on a large number of bacterial genomes. The source code of our method can be freely downloaded at https://github.com/maqin2001/PromotePredictor.

Genome Wide Measurements of the Sequence Dependence of Nucleosomal DNA Flexibility

The physical properties of DNA such as bending flexibility are sequence dependent. However, how DNA flexibility varies across the genome, how sequence mutations affect downstream biological processes by changing DNA flexibility, and how DNA flexibility may have contributed to the evolutionary selection of DNA sequences, are questions that have rarely been explored. By combining single molecule techniques with next generation sequencing, we have developed an assay to create a genome wide map of DNA flexibility. An input library of tens of thousands of short DNA fragment is end modified to permit cyclization. A selection condition is imposed, whereby stiffer molecules that fail to cyclize are preferentially digested. The surviving molecules are sequenced and ranked in flexibility according to their survival probability. We have applied this technique to study the flexibility of DNA at ten thousand nucleosomal locations in Saccharomyces cerevisiae. We find that nucleosomal occupancy correlates with local DNA flexibility, suggesting that DNA flexibility has played a role in the evolution of nucleosomal sequences. Correlations between DNA flexibility asymmetry and histone occupancy asymmetry within nucleosomes suggest that in vivo, nucleosomes exist in partially unraveled states, and DNA is under tension. Finally, our data shows that DNA flexibility patterns on the first few nucleosomes downstream of transcription start sites may have evolved to facilitate initiation of transcription, and is a predictor of gene expression levels. A detailed understanding of how DNA sequences, DNA chemical modifications, and DNA:protein interactions affect downstream biological processes by altering local DNA flexibility, will allow us to appreciate how physical forces have shaped the evolution of genomes.

Designed Transcriptional Regulation in Mammalian Cells Based on TALE- and CRISPR/dCas9.

Transcriptional regulation lies at the center of many cellular processes and is the result of cellular response to different external and internal signals. Control of transcription of selected genes enables an unprecedented access to shape the cellular response. While orthogonal transcription factors from bacteria, yeast, plants, or other cells have been used to introduce new cellular logic into mammalian cells, the discovery of designable modular DNA binding domains, such as Transcription Activator-Like Effectors (TALEs) and the CRISPR system, enable targeting of almost any selected DNA sequence. Fusion or conditional association of DNA targeting domain with transcriptional effector domains enables controlled regulation of almost any endogenous or ectopic gene. Moreover, the designed regulators can be linked into genetic circuits to implement complex responses, such as different types of Boolean functions and switches. In this chapter, we describe the protocols for achieving efficient transcriptional regulation with TALE- and CRISPR-based designed transcription factors in mammalian cells.