DNA Sequence Recognition Research Articles

Allele-specific transcription factor binding across human brain regions offers mechanistic insight into eQTLs.

Transcription factors (TFs) regulate gene expression by facilitating or disrupting the formation of transcription initiation machinery at particular genomic loci. Because TF occupancy is driven in part by recognition of DNA sequence, genetic variation can influence TF-DNA associations and gene regulation. To identify variants that impact TF binding in human brain tissues, we assessed allele-specific binding (ASB) at heterozygous variants for 94 TFs in nine brain regions from two donors. Leveraging graph genomes constructed from phased genomic sequence data, we compared ChIP-seq signals between alleles at heterozygous variants within each brain region and identified thousands of variants exhibiting ASB for at least one TF. ASB reproducibility was measured by comparisons between independent experiments both within and between donors. We found that rare alleles in the general population more frequently led to reduced TF binding, whereas common alleles had an equal likelihood of increasing or decreasing binding. Further, for ASB variants in predicted binding motifs, the favored allele tended to be the one with the stronger expected motif match, but this concordance was not observed within highly occupied sites. We also found that neuron-specific cis-regulatory elements (cCREs), in contrast with oligodendrocyte-specific cCREs, showed depletion of ASB variants. We identified 2670 ASB variants associated with evidence for allele-specific gene expression in the brain from GTEx data and observed increasing eQTL effect direction concordance as ASB significance increases. These results provide a valuable and unique resource for mechanistic analysis of cis-regulatory variation in human brain tissue.

Burkholderia cenocepacia epigenetic regulator M.BceJIV simultaneously engages two DNA recognition sequences for methylation

Burkholderia cenocepacia is an opportunistic and infective bacterium containing an orphan DNA methyltransferase called M.BceJIV with roles in regulating gene expression and motility of the bacterium. M.BceJIV recognizes a GTWWAC motif (where W can be an adenine or a thymine) and methylates N6 of the adenine at the fifth base position. Here, we present crystal structures of M.BceJIV/DNA/sinefungin ternary complex and allied biochemical, computational, and thermodynamic analyses. Remarkably, the structures show not one, but two DNA substrates bound to the M.BceJIV dimer, with each monomer contributing to the recognition of two recognition sequences. We also show that methylation at the two recognition sequences occurs independently, and that the GTWWAC motifs are enriched in intergenic regions in the genomes of B. cenocepacia strains. We further computationally assess the interactions underlying the affinities of different ligands (SAM, SAH, and sinefungin) for M.BceJIV, as a step towards developing selective inhibitors for limiting B. cenocepacia infection.

The Dynamic Plasticity of P. aeruginosa CueR Copper Transcription Factor upon Cofactor and DNA Binding.

Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram-negative bacterial copper regulator. The structure of E. coli CueR complexed with Cu(I) and DNA was published, since then many studies have shed light on its function. However, P. aeruginosa CueR, which shows high sequence similarity to E. coli CueR, has been less studied. Here, we applied room-temperature electron paramagnetic resonance (EPR) measurements to explore changes in dynamics of P. aeruginosa CueR in dependency of copper concentrations and interaction with two different DNA promoter regions. We showed that P. aeruginosa CueR is less dynamic than the E. coli CueR protein and exhibits much higher sensitivity to DNA binding as compared to its E. coli CueR homolog. Moreover, a difference in dynamical behavior was observed when P. aeruginosa CueR binds to the copZ2 DNA promoter sequence compared to the mexPQ-opmE promoter sequence. Such dynamical differences may affect the expression levels of CopZ2 and MexPQ-OpmE proteins in P. aeruginosa. Overall, such comparative measurements of protein-DNA complexes derived from different bacterial systems reveal insights about how structural and dynamical differences between two highly homologous proteins lead to quite different DNA sequence-recognition and mechanistic properties.

Re-engineered guide RNA enables DNA loops and contacts modulating repression in E. coli.

RNA serves as information media as well as molecular scaffold in nature and synthetic systems. The single guide RNA (sgRNA) widely applied in CRISPR techniques exemplifies both functions, with a guide region bearing DNA base-pairing information, and a structural motif for Cas9 protein scaffolding. The scaffold region has been modified by fusing RNA aptamers to the tetra-stem loop. The guide region is typically not regarded as a pluggable module as it encodes the essential function of DNA sequence recognition. Here, we investigate a chimera of two sgRNAs, with distinct guide sequences joined by an RNA linker (dgRNA), regarding its DNA binding function and loop induction capability. First, we studied the sequence bi-specificity of the dgRNA and discovered that the RNA linker allows distal parts of double-stranded DNA to be brought into proximity. To test the activity of the dgRNA in organisms, we used the LacZ gene as a reporter and recapitulated the loop-mediated gene inhibition by LacI in E. coli. We found that the dgRNA can be applied to target distal genomic regions with comparable levels of inhibition. The capability of dgRNA to induce DNA contacts solely requires dCas9 and RNA, making it a minimal system to remodel chromosomal conformation in various organisms.

The transcriptional repressor B cell lymphoma 6 regulates CXCR3 chemokine and human leukocyte antigen II expression in endothelial cells

Interferon gamma (IFN-γ) induces an endothelial proimmunogenic phenotype through the JAK/STAT1 pathway, which can shape the activation of alloreactive leukocytes in transplant rejection. In immune cells, the DNA-binding protein B cell lymphoma 6 (BCL6) controls the transcription of inflammatory genes. This study tested if BCL6 modulates IFN-γ-induced gene expression in endothelial cells. In vitro, BCL6 was IFN-γ-inducible in primary human endothelium, along with CXCR3 chemokines and human leukocyte antigen (HLA). BCL6, HLA II, and CXCL9 were also increased in human cardiac transplants during acute rejection. Knockdown of BCL6 augmented, whereas overexpression and BTB domain inhibitors (BCL6-BTBi) suppressed, HLA II and CXCR3 chemokine expression but not HLA I. Further, BCL6 had a greater effect on HLA-DR and DP but was less involved in regulating HLA-DQ expression. The effect correlated with BCL6 binding motifs in or near affected genes. The BCL6 DNA recognition sequence was highly similar to that of STAT1, and BTBi reduced STAT1’s transcriptional activity in vitro. Our results show for the first time that BCL6 selectively controls IFN-γ-induced endothelial gene expression, advancing our understanding of the endogenous mechanisms regulating donor immunogenicity.

Integration of a Cas12a-mediated DNAzyme actuator with efficient RNA extraction for ultrasensitive colorimetric detection of viral RNA

Developing highly sensitive and specific on-site tests is imperative to strengthen preparedness against future emerging infectious diseases. Here, we describe the construction of a Cas12a-mediated DNAzyme actuator capable of converting the recognition of a specific DNA sequence into an amplified colorimetric signal. To address viral RNA extraction challenges for on-site applications, we developed a rapid and efficient method capable of lysing the viral particles, preserving the released viral RNA, and concentrating the viral RNA. Integration of the DNAzyme actuator with the viral RNA extraction method and loop-mediated isothermal amplification enables a streamlined colorimetric assay for highly sensitive colorimetric detection of respiratory RNA viruses in gargle and saliva. This assay can detect as few as 83 viral particles/100 μL in gargle and 166 viral particles/100 μL in saliva. The entire assay, from sample processing to visual detection, was completed within 1 h at a single controlled temperature. We validated the assay by detecting SARS-CoV-2 in 207 gargle and saliva samples, achieving a clinical sensitivity of 96.3 % and specificity of 100%. The assay is adaptable for detecting specific nucleic acid sequences in other pathogens and is suitable for resource-limited settings.

Structural basis for specific DNA sequence recognition by the transcription factor NFIL3

The CCAAT/enhancer-binding proteins (C/EBPs) constitute a family of pivotal transcription factors involved in tissue development, cellular function, proliferation, and differentiation. NFIL3, as one of them, plays an important role in regulating immune cell differentiation, circadian clock system and neural regeneration, yet its specific DNA recognition mechanism remains enigmatic. In this study, we showed by the ITC binding experiments that NFIL3 prefers to bind to the TTACGTAA DNA motif. Our structural studies revealed that the α-helical NFIL3 bZIP domain dimerizes through its leucine zipper region, and binds to DNA via its basic region. The two basic regions of the NFIL3 bZIP dimer were pushed apart upon binding to DNA, facilitating the snug accommodation of the two basic regions within the major grooves of the DNA. Remarkably, our binding and structural data also revealed that both NFIL3 and C/EBPα/β demonstrated a shared preference for the TTACGTAA sequence. Furthermore, our study revealed that disease-associated mutations within the NFIL3 bZIP domain resulted in either reduction or complete disruption of its DNA binding ability. These discoveries not only provide valuable insights into the DNA binding mechanisms of NFIL3 but also elucidate the causal role of NFIL3 mutations in disease pathogenesis.

DNA Sequence Control of Enzyme Filamentation and Activation of the SgrAI Endonuclease.

Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, Streptomyces griseus, is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.

Molecular mechanism of specific DNA sequence recognition by NRF1.

Nuclear respiratory factor 1 (NRF1) regulates the expression of genes that are vital for mitochondrial biogenesis, respiration, and various other cellular processes. While NRF1 has been reported to bind specifically to GC-rich promoters as a homodimer, the precise molecular mechanism governing its recognition of target gene promoters has remained elusive. To unravel the recognition mechanism, we have determined the crystal structure of the NRF1 homodimer bound to an ATGCGCATGCGCAT dsDNA. In this complex, NRF1 utilizes a flexible linker to connect its dimerization domain (DD) and DNA binding domain (DBD). This configuration allows one NRF1 monomer to adopt a U-turn conformation, facilitating the homodimer to specifically bind to the two TGCGC motifs in the GCGCATGCGC consensus sequence from opposite directions. Strikingly, while the NRF1 DBD alone could also bind to the half-site (TGCGC) DNA of the consensus sequence, the cooperativity between DD and DBD is essential for the binding of the intact GCGCATGCGC sequence and the transcriptional activity of NRF1. Taken together, our results elucidate the molecular mechanism by which NRF1 recognizes specific DNA sequences in the promoters to regulate gene expression.

Correlative analysis of transcriptome and proteome in Penaeus vannamei reveals key signaling pathways are involved in IFN-like antiviral regulation mediated by interferon regulatory factor (PvIRF)

Interferon regulatory factors (IRFs) are crucial transcription factors that regulate interferon (IFN) induction in response to pathogen invasion. The regulatory mechanism of IRF has been well studied in vertebrates, but little has been known in arthropods. Therefore, in order to obtain new insights into the potential molecular mechanism of Peneaus vannamei IRF (PvIRF) in response to viral infection, comprehensive comparative analysis of the transcriptome and proteome profiles in shrimp infected with WSSV after knocking down PvIRF was conducted by using RNA sequencing (RNA-seq) and isobaric tags for relative and absolute quantification (iTRAQ). The sequence characterization, molecular functional evolution and 3D spatial structure of PvIRF were analyzed by using bioinformatics methods. PvIRF share the higher homology with different species in N-terminal end (containing DNA binding domain (DBD) including DNA sequence recognition sites and metal binding site) than that in C-terminal end. Within 4 IRF subfamilies of vertebrates, PvIRF had closer relationship with IRF1 subfamily. The DBD of PvIRF and C. gigas IRF1a were composed of α-helices and β-folds which was similar with the DBD structure of M. musculus IRF2. Interestingly, different from the five Tryptophan repeats highly homologous in the DBD of vertebrate IRF, the first and fifth tryptophans of PvIRF mutate to Phenylalanine and Leucine respectively, while the mutations were conserved among shrimp IRFs. RNAi knockdown of PvIRF gene by double-strand RNA could obviously promote the in vivo propagation of WSSV in shrimp and increase the mortality of WSSV-infected shrimp. It suggested that PvIRF was involved in inhibiting the replication of WSSV in shrimp. A total of 8787 transcripts and 2846 proteins were identified with significantly differential abundances in WSSV-infected shrimp after PvIRF knockdown, among which several immune-related members were identified and categorized into 10 groups according to their possible functions. Furthermore, the variation of expression profile from members of key signaling pathways involving JAK/STAT and Toll signaling pathway implied that they might participate IRF-mediated IFN-like regulation in shrimp. Correlative analyses indicated that 722 differentially expressed proteins (DEPs) shared the same expression profiles with their corresponding transcripts, including recognition-related proteins (CTLs and ITGs), chitin-binding proteins (peritrophin), and effectors (ALFs and SWD), while 401 DEPs with the opposite expression profiles across the two levels emphasized the critical role of post-transcriptional and post-translational modification. The results provide candidate signaling pathway including pivotal genes and proteins involved in the regulatory mechanism of interferon mediated by IRF on shrimp antiviral response. This is the first report in crustacean to explore the IFN-like antiviral regulation pathway mediated by IRF on the basis of transcriptome and proteomics correlative analysis, and will provide new ideas for further research on innate immune and defense mechanisms of crustacean.

Whole-genome detection using multivalent DNA-coated colloids

To minimize the incorrect use of antibiotics, there is a great need for rapid and inexpensive tests to identify the pathogens that cause an infection. The gold standard of pathogen identification is based on the recognition of DNA sequences that are unique for a given pathogen. Here, we propose and test a strategy to develop simple, fast, and highly sensitive biosensors that make use of multivalency. Our approach uses DNA-functionalized polystyrene colloids that distinguish pathogens on the basis of the frequency of selected short DNA sequences in their genome. Importantly, our method uses entire genomes and does not require nucleic acid amplification. Polystyrene colloids grafted with specially designed surface DNA probes can bind cooperatively to frequently repeated sequences along the entire genome of the target bacteria, resulting in the formation of large and easily detectable colloidal aggregates. Our detection strategy allows "mix and read" detection of the target analyte; it is robust and highly sensitive over a wide concentration range covering, in the case of our test target genome Escherichia coli bl21-de3, 10 orders of magnitude from [Formula: see text] to [Formula: see text] copies/mL. The sensitivity compares well with state-of-the-art sensing techniques and has excellent specificity against nontarget bacteria. When applied to real samples, the proposed technique shows an excellent recovery rate. Our detection strategy opens the way to developing a robust platform for pathogen detection in the fields of food safety, disease control, and environmental monitoring.

Synthesis and Evaluation of MGB Polyamide-Oligonucleotide Conjugates as Gene Expression Control Compounds.

MGB polyamide-oligonucleotide conjugates ON 1-4 with linked MGB polyamides at the 2-exocyclic amino group of a guanine base using aminoalkyl linkers were synthesized and evaluated in terms of binding affinity for complementary DNA containing the MGB polyamide binding sequence using T m and CD analyses. The MGB polyamides comprised pyrrole polyamides (Py4- and Py3-), which possess binding affinity for A-T base pairs, and imidazole (Im3-) and pyrrole-γ-imidazole (Py3-γ-Im3-) polyamide hairpin motifs, which possess binding affinity for C-G base pairs. It was found that the stability of modified dsDNA was greatly influenced by the linker length. Py4- and Py3-oligonucleotide conjugates (ON 1 (n = 4) and ON 2 (n = 4)) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA. Although Im3-oligonucleotide conjugate ON 3 (n = 4) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA, stabilization of dsDNA by the imidazole amide moiety of ON 3 (n = 4) was lower compared with the pyrrole amide moiety of ON 2 (n = 4). The Py3-γ-Im3-oligonucleotide conjugate ON 4 (n = 2), which possesses binding affinity for C-G base pairs via a pyrrole/imidazole combination and contains a 2-aminoethyl linker, showed high binding ability for complementary DNA. Furthermore, the DNA sequence recognition of MGB polyamide-oligonucleotide conjugates was investigated using single-base mismatch DNAs, which possess a mismatch base in the MGB polyamide binding sequence. The Py3-γ-Im3-oligonucleotide conjugate ON 4 (n = 2) showed high sequence recognition ability for complementary DNA.

DNA Methylation and RNA-DNA Hybrids Regulate the Single-Molecule Localization of a DNA Methyltransferase on the Bacterial Nucleoid.

Bacterial DNA methyltransferases (MTases) function in restriction modification systems, cell cycle control, and the regulation of gene expression. DnmA is a recently described DNA MTase that forms N6-methyladenosine at nonpalindromic 5'-GACGAG-3' sites in Bacillus subtilis, yet how DnmA activity is regulated is unknown. To address DnmA regulation, we tested substrate binding in vitro and found that DnmA binds poorly to methylated DNA and to an RNA-DNA hybrid with the DNA recognition sequence. Further, DnmA variants with amino acid substitutions that disrupt cognate sequence recognition or catalysis also bind poorly to DNA. Using superresolution fluorescence microscopy and single-molecule tracking of DnmA-PAmCherry, we characterized the subcellular DnmA diffusion and detected its preferential localization to the replisome region and the nucleoid. Under conditions where the chromosome is highly methylated, upon RNA-DNA hybrid accumulation, or with a DnmA variant with severely limited DNA binding activity, DnmA is excluded from the nucleoid, demonstrating that prior methylation or accumulation of RNA-DNA hybrids regulates the association of DnmA with the chromosome in vivo. Furthermore, despite the high percentage of methylated recognition sites and the proximity to putative endonuclease genes conserved across bacterial species, we find that DnmA fails to protect B. subtilis against phage predation, suggesting that DnmA is functionally an orphan MTase involved in regulating gene expression. Our work explores the regulation of a bacterial DNA MTase and identifies prior methylation and RNA-DNA hybrids as regulators of MTase localization. These MTase regulatory features could be common across biology. IMPORTANCE DNA methyltransferases (MTases) influence gene expression, cell cycle control, and host defense through DNA modification. Predicted MTases are pervasive across bacterial genomes, but the vast majority remain uncharacterized. Here, we show that in the soil microorganism Bacillus subtilis, the DNA MTase dnmA and neighboring genes are remnants of a phage defense system that no longer protects against phage predation. This result suggests that portions of the bacterial methylome may originate from inactive restriction modification systems that have maintained methylation activity. Analysis of DnmA movement in vivo shows that active DnmA localizes in the nucleoid, suggesting that DnmA can search for recognition sequences throughout the nucleoid region with some preference for the replisome. Our results further show that prior DNA methylation and RNA-DNA hybrids regulate DnmA dynamics and nucleoid localization, providing new insight into how DNA methylation is coordinated within the cellular environment.

Zinc controls operator affinity of human transcription factor YY1 by mediating dimerization via its N-terminal region

Human protein Yin Yang 1 (YY1) controls the transcription of hundreds of genes both positively and negatively through interactions with a wide range of partner proteins. Results presented here from proteolytic sensitivity, calorimetry, circular dichroism, fluorescence, NMR, size-exclusion chromatography, SELEX, and EMSA show that purified YY1 forms dimers via its disordered N-terminal region with strong zinc-ion concentration dependence. The YY1 dimer is shown to bind tandem repeats of a canonical recognition DNA sequence with high affinity, and analysis of human YY1 regulatory sites shows that many contain repeats of its recognition elements. YY1 dimerization may compete with partner protein interactions, making control by zinc ion concentration a previously unrecognized factor affecting YY1 gene regulation. Indeed, YY1 is known to be important in many pathogenic processes, including neoplasia, in which zinc ion concentrations are altered. The present results incentivize studies in vivo or in vitro that explore the role of zinc ion concentration in YY1-mediated gene expression.

A 5'-Flanking C/G Pair at the Core Region Enhances the Recognition and Binding of Kaiso to Methylated DNA.

Methyl CpG binding proteins (MBPs) are transcription factors that recognize the methylated CpG sites in DNA and mediate the DNA methylation signal into various downstream cellular processes. The C2H2 zinc finger (ZF) protein, Kaiso, also an MBP, preferentially binds to two symmetrically methylated CpG sites in DNA sequences via C-terminal C2H2 ZF domains and mediates the transcription regulation process. Investigation of the molecular mechanism of the recognition of methylated DNA (meDNA) by Kaiso is important to understand how this protein reads and translates this methylation signal into downstream transcription outcomes. Despite previous studies in Kaiso-meDNA interactions, detailed structural investigations on the sequence-specific interaction of Kaiso with the meDNA sequence are still lacking. In this work, we used molecular modeling and molecular dynamics (MD) simulation-based computational approaches to investigate the recognition of various methylated DNA sequences by Kaiso. Our MD simulation results show that the Kaiso-meDNA interaction is sequence specific. The recognition of meDNA by Kaiso is enhanced in the MeECad sequence compared to the MeCG2 sequence. Compared to the 5'-flanking T/A pair in MeCG2, both MeCG2_mutCG and MeECad sequences show that a C/G base pair allows GLU535 of Kaiso to preferably recognize and bind the core mCpG site. The core mCGmCG site is crucial for the recognition process and formation of a stable complex. Our results reveal that the 5'-flanking nucleotides are also important for the enhanced binding and recognition of methylated sites.

Hoogsteen Base Pairings

In contrast to Watson-Crick (WC) base pairing, Hoogsteen (HG) base pairing involves flipping a purine base 180° between its anti and syn conformation. Recent studies have shown that HG pairs coexist in dynamical equilibrium, and several biological functions depend on them. This significance has stirred computational research on this base-pairing transition. However, a methodical reproduction of sequence variations has continued to be out of reach. It is becoming increasingly clear that Hoogsteen base pairs play a crucial role in DNA replication, recognition, damage repair, and incorrect sequence repair. The Protein Data Bank contains a variety of Hoogsteen base pairing modes that include the preference for A–T versus G–C bps, TA versus GG steps, and a preference for 5'-purines at terminal ends. RNA A-form duplexes are strongly disfavored by Hoogsteen base pairs, in stark contrast to B-form DNA. Therefore, N1-methyl adenosine and N1-methyl guanosine, which transpire in DNA as alkylation impairment and in RNA as posttranscriptional adjustments, have great differences in effects. They create G–C+ and A–U Hoogsteen base pairs in duplex DNA that preserve the structural integrity of the double helix, but obstruct base pairing altogether and induce local duplex melting in RNA, providing a mechanism for potently disrupting RNA structure through posttranscriptional modifications. In duplex DNA, they maintain the structural integrity of the double helix by creating G–C+ and A–U Hoogsteen base pairs, but block base pairing altogether and cause local duplex melting in RNA, thus providing a potent means for disrupting RNA structure post transcriptionally. As a result of the markedly different inclinations for B-DNA and A-RNA to form Hoogsteen base pairs, they may be able to balance the opposing demands of maintaining genome stability and dynamically modulating the epitranscriptome. This review examines the occurrence of Hoogsteen base pairs in DNA and RNA duplexes.

Laser-Induced graphene electrodes for highly sensitive detection of DNA hybridization via consecutive cytosines (polyC)-DNA-based electrochemical biosensors

Numerous carbon-based biosensors issued mechanical exfoliation, epitaxial growth, reduced graphene oxide, and chemical vapor deposition have been investigated for highly sensitive and specific detection of DNA. As a promising route for designing electrochemical biosensor-based flexible substrates, the laser-induced graphene technique, which provides a cheap, technologically simple, and highly robust sensing platform, has been widely adopted. However, DNA-based biosensors' efficiency is strongly dependent on how DNA probes are tethered to the nanomaterials. In view of this, poly-cytosine (poly-C) DNA has shown outstanding adsorption to multiple inorganic nanomaterials, including gold (Au), zinc oxide (ZnO), tungsten disulfide (WS2), graphene oxide (GO), and graphene. In this work, a poly C(15)-tailed diblock DNA probe is used to anchor to carbonized working electrode issued laser-induced method. Meanwhile, the second block modified with ferrocene (Fc) derivatives is lifted at the surface for DNA sequence recognition. Following this strategy, the developed biosensor leads to a limit of detection (LOD) of 57 fM, which was superior or comparable to some previously reported methods. Moreover, the proposed electrochemical DNA biosensor exhibits high specificity in differentiating the complementary DNA from non-complementary DNA (ncDNA), and mismatched DNAs (MM-DNA) sequences. Finally, the easily constructed laser-induced graphene electrode biosensor showed an ability to detect DNA in human serum as a complex environment, making our approach a promising avenue for disease diagnosis.

Choosing the right partner in hormone-dependent gene regulation: Glucocorticoid and progesterone receptors crosstalk in breast cancer cells

Steroid hormone receptors (SHRs) belong to a large family of ligand-activated nuclear receptors that share certain characteristics and possess others that make them unique. It was thought for many years that the specificity of hormone response lay in the ligand. Although this may be true for pure agonists, the natural ligands as progesterone, corticosterone and cortisol present a broader effect by simultaneous activation of several SHRs. Moreover, SHRs share structural and functional characteristics that range from similarities between ligand-binding pockets to recognition of specific DNA sequences. These properties are clearly evident in progesterone (PR) and glucocorticoid receptors (GR); however, the biological responses triggered by each receptor in the presence of its ligand are different, and in some cases, even opposite. Thus, what confers the specificity of response to a given receptor is a long-standing topic of discussion that has not yet been unveiled. The levels of expression of each receptor, the differential interaction with coregulators, the chromatin accessibility as well as the DNA sequence of the target regions in the genome, are reliable sources of variability in hormone action that could explain the results obtained so far. Yet, to add further complexity to this scenario, it has been described that receptors can form heterocomplexes which can either compromise or potentiate the respective hormone-activated pathways with its possible impact on the pathological condition. In the present review, we summarized the state of the art of the functional cross-talk between PR and GR in breast cancer cells and we also discussed new paradigms of specificity in hormone action.

Flexibility of flanking DNA is a key determinant of transcription factor affinity for the core motif

Selective gene regulation is mediated by recognition of specific DNA sequences by transcription factors (TFs). The extremely challenging task of searching out specific cognate DNA binding sites among several million putative sites within the eukaryotic genome is achieved by complex molecular recognition mechanisms. Elements of this recognition code include the core binding sequence, the flanking sequence context, and the shape and conformational flexibility of the composite binding site. To unravel the extent to which DNA flexibility modulates TF binding, in this study, we employed experimentally guided molecular dynamics simulations of ternary complex of closely related Hox heterodimers Exd-Ubx and Exd-Scr with DNA. Results demonstrate that flexibility signatures embedded in the flanking sequences impact TF binding at the cognate binding site. A DNA sequence has intrinsic shape and flexibility features. While shape features are localized, our analyses reveal that flexibility features of the flanking sequences percolate several basepairs and allosterically modulate TF binding at the core. We also show that lack of flexibility in the motif context can render the cognate site resistant to protein-induced shape changes and subsequently lower TF binding affinity. Overall, this study suggests that flexibility-guided DNA shape, and not merely the static shape, is a key unexplored component of the complex DNA-TF recognition code.

Conformation impact in the deformation of DNA TATA-box

The ability to undergo specific deformation in response to conformational changes of some sites of the macromolecule is the key element of DNA genetic activity regulation in biological cells. The deformation of the definite sequences in DNA chain provides an accurate reading of genetic information and the process of protein synthesis passing. The model based on the analysis of conformational changes of the TATA nucleotide sequence in DNA double helix is developed. In addition to elastic components (bending, twisting), the presented model includes the conformational rearrangements of the TATA-box. Obtained form, value, and energy of DNA cite deformation allow to offer the probable mechanism of recognition of key DNA sequences and makes it possible to explain the high accuracy of the processes of reproducing of genetic information in biological cells. The formulated physical mechanism of DNA deformation can be one of the main principles of genetic information realization.