Heterogeneous nuclear ribonucleoprotein E1 binds polycytosine DNA and monitors genome integrity.

Heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) is a tumor suppressor protein that binds site- and structure-specifically to RNA sequences to regulate mRNA stability, facilitate alternative splicing, and suppress protein translation on several metastasis-associated mRNAs. Here, we show that hnRNP E1 binds polycytosine-rich DNA tracts present throughout the genome, including those at promoters of several oncogenes and telomeres and monitors genome integrity. It binds DNA in a site- and structure-specific manner. hnRNP E1-knockdown cells displayed increased DNA damage signals including γ-H2AX at its binding sites and also showed increased mutations. UV and hydroxyurea treatment of hnRNP E1-knockdown cells exacerbated the basal DNA damage signals with increased cell cycle arrest, activation of checkpoint proteins, and monoubiquitination of proliferating cell nuclear antigen despite no changes in deubiquitinating enzymes. DNA damage caused by genotoxin treatment localized to hnRNP E1 binding sites. Our work suggests that hnRNP E1 facilitates functions of DNA integrity proteins at polycytosine tracts and monitors DNA integrity at these sites.

DNA Translocation through Vertically Stacked 2D Layers of Graphene and Hexagonal Boron Nitride Heterostructure Nanopore.

Cost-effective, fast, and reliable DNA sequencing can be enabled by advances in nanopore-based methods, such as the use of atomically thin graphene membranes. However, strong interaction of DNA bases with graphene leads to undesirable effects such as sticking of DNA strands to the membrane surface. While surface functionalization is one way to counter this problem, here, we present another solution based on a heterostructure nanopore system, consisting of a monolayer of graphene and hexagonal boron nitride (hBN) each. Molecular dynamics studies of DNA translocation through this heterostructure nanopore revealed a surprising and crucial influence of the heterostructure layer order in controlling the base specific signal variability. Specifically, the heterostructure with graphene on top of hBN had nearly 3-10× lower signal variability than the one with hBN on top of graphene. Simulations point to the role of differential underside sticking of DNA bases as a possible reason for the observed influence of the layer order. Our studies can guide the development of experimental systems to study and exploit DNA translocation through two-dimensional heterostructure nanopores for single molecule sequencing and sensing applications.

Hysteresis in poly-2'-deoxycytidine i-motif folding is impacted by the method of analysis as well as loop and stem lengths.

In DNA, i-motif (iM) folds occur under slightly acidic conditions when sequences rich in 2'-deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pHT ) between the folded and unfolded states. Two different experiments to determine pHT values via circular dichroism (CD) spectroscopy were performed on poly-dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pHT values were dependent on the titration experiment performed, and (2) pH-induced denaturing or annealing processes produced isothermal hysteresis in the pHT values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly-dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly-dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pHT parameter measured, and hysteresis was observed in the pHT and Tm values.

Flexibility and Preorganization of Fluorescent Nucleobase-Pyrene Conjugates Control DNA and RNA Recognition.

We synthesized a new amino acid-fluorescent nucleobase derivative (qAN1-AA) and from it two new fluorescent nucleobase-fluorophore (pyrene) conjugates, whereby only the analogue with the longer and more flexible linker (qAN1-pyr2) self-folded into intramolecularly stacked qAN1/pyrene conformation, yielding characteristic, 100 nm-red-shifted emission (λmax = 500 nm). On the contrary, the shorter and more rigid linker resulted in non-stacked conformation (qAN1-pyr1), characterized by the emission of free pyrene at λmax = 400 nm. Both fluorescent nucleobase-fluorophore (pyrene) conjugates strongly interacted with ds-DNA/RNA grooves with similar affinity but opposite fluorescence response (due to pre-organization), whereas the amino acid-fluorescent base derivative (qAN1-AA) was inactive. However, only intramolecularly self-folded qAN1-pyr2 showed strong fluorescence selectivity toward poly U (Watson-Crick complementary to qAN1 nucleobase) and poly A (reverse Hoogsteen complementary to qAN1 nucleobase), while an opposite emission change was observed for non-complementary poly G and poly C. Non-folded analogue (qAN1-pyr1) showed no ss-RNA selectivity, demonstrating the importance of nucleobase-fluorophore pre-organization.

Insights on the interaction of phenothiazinium dyes methylene blue and new methylene blue with synthetic duplex RNAs through spectroscopy and modeling.

The ubiquitous influence of double stranded RNAs in biological events makes them imperative to gather data based on specific binding procedure of small molecules to various RNA conformations. Particular interest may be attributed to situations wherein small molecules target RNAs altering their structures and causing functional modifications. The main focus of this study is to delve into the interactive pattern of two small molecule phenothiazinium dyes, methylene blue and new methylene blue, with three duplex RNA polynucleotides-poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C) by spectroscopic and molecular modeling techniques. Analysis of data as per Scatchard and Benesi-Hildebrand methodologies revealed highest affinity of these dyes to poly(A).poly(U) and least to poly(I).poly(C). In addition to fluorescence quenching, viscometric studies also substantiated that the dyes follow different modes of binding to different RNA polynucleotides. Distortion in the RNA structures with induced optical activity in the otherwise optically inactive dye molecules was evidenced from circular dichroism results. Dye-induced RNA structural modification occurred from extended conformation to compact particles visualized by atomic force microscopy. Molecular docking results revealed different binding patterns of the dye molecules within the RNA duplexes. The novelty of the present work lies towards a new contribution of the phenothiazinium dyes in dysfunctioning double stranded RNAs, advancing our knowledge to their potential use as RNA targeted small molecules.

Temperature Dependence of the STM Morphology and Electronic Level Structure of Silver-Containing DNA.

Understanding the effect of external conditions, temperature in particular, on novel nanomaterials is of great significance. The powerful ability of scanning tunneling microscopy (STM) to characterize topography and electronic levels on a single molecule scale is utilized herein to characterize individual silver-containing poly(dG)-poly(dC) DNA molecules, at different temperatures. These measurements indicate that the molecule is a truly hybrid metal-organic nanomaterial with electronic states originating from both the DNA and the embedded silver. The temperature dependence of this density of states (DOS) leads to the temperature dependent STM apparent height of the molecule-a phenomenon that has not been observed before for other complex nanostructures.

Scanning Tunneling Microscopy and Spectroscopy of Novel Silver-Containing DNA Molecules.

The quest for a suitable molecule to pave the way to molecular nanoelectronics has been met with obstacles for over a decade. Candidate molecules such as carbon nanotubes lack the appealing trait of self-assembly, while DNA seems to lack the desirable feature of conductivity. Silver-containing poly(dG)-poly(dC) DNA (E-DNA) molecules have recently been reported as promising candidates for molecular electronics, owing to the selectivity of their metallization, their thin and uniform structure, their resistance to deformation, and their maximum possible high conductivity. Ultrahigh vacuum (UHV) scanning tunneling microscopy (STM) of E-DNA presents an elaborate high-resolution morphology characterization of these unique molecules, along with a detailed depiction of their electronic level structure. The energy levels found for E-DNA indicate a novel truly hybrid metal-molecule structure, potentially more conductive than other DNA-based alternatives.

Formation of i-motifs from acyclic (l)-threoninol nucleic acids.

Acyclic (l)-threoninol nucleic acids ((l)-aTNA) containing poly-cytosines are prepared and investigated at various pH values, revealing the formation of a highly stable structure at lower pH that have the characteristics of an i-motif. Depending on the sequence, the aTNA forms inter-, bi- and intra-molecular i-motif structures. Pyrene was conjugated to aTNA sequences and both monomeric and excimer fluorescence were efficiently quenched by the i-motif structures and thus demonstrated that the aTNA i-motif can serve as a pH switch.

Structural transitions in poly(A), poly(C), poly(U), and poly(G) and their possible biological roles.

The homopolynucleotide (homo-oligonucleotide) tracts function as regulatory elements at various stages of mRNAs life cycle. Numerous cellular proteins specifically bind to these tracts. Among them are the different poly(A)-binding proteins, poly(C)-binding proteins, multifunctional fragile X mental retardation protein which binds specifically both to poly(G) and poly(U) and others. Molecular mechanisms of regulation of gene expression mediated by homopolynucleotide tracts in RNAs are not fully understood and the structural diversity of these tracts can contribute substantially to this regulation. This review summarizes current knowledge on different forms of homoribopolynucleotides, in particular, neutral and acidic forms of poly(A) and poly(C), and also biological relevance of homoribopolynucleotide (homoribo-oligonucleotide) tracts is discussed. Under physiological conditions, the acidic forms of poly(A) and poly(C) can be induced by proton transfer from acidic amino acids of proteins to adenine and cytosine bases. Finally, we present potential mechanisms for the regulation of some biological processes through the formation of intramolecular poly(A) duplexes.

Affinity-Modulated Molecular Beacons on MoS2 Nanosheets for MicroRNA Detection.

DNA-functionalized layered two-dimensional transition-metal dichalcogenides have attracted tremendous interest for constructing biosensors in recent years. In this work, we report diblock molecular beacons with poly-cytosine (polyC) tails anchored on molybdenum disulfide (MoS2) nanosheets as probes for microRNA detection. The polyC block is adsorbed on MoS2 and the molecular beacon block is available for hybridization to the target; duplex-specific nuclease provides signal amplification by target recycling. By changing the length of polyC, we regulate the density of probes on MoS2 and inhibit the adsorption of enzyme-cleaved oligonucleotides, thereby leading to higher quenching efficiency. PolyC-mediated molecular beacons on MoS2 have very low background signal, ultrahigh sensitivity (limit of detection ∼3.4 fM), specificity to detect a single nucleotide mismatch, and selectivity to detect target microRNA from serum samples. This detection platform holds great potential for quantitative analysis of miRNAs in clinical diagnosis and biomedical research.

Novel methods for nucleotide length control in double-stranded polyinosinic-polycytidylic acid production using uneven length components.

Polyinosinic-polycytidylic acid (PIC), a double-stranded RNA that induces innate immunity in mammals, is a candidate immunopotentiator for pharmaceuticals. The potency and adverse effects of PIC are strongly correlated with the nucleotide length, and the inability to precisely control the length in PIC production limits its practical use. Length extension during the annealing process is the major factor underlying the lack of control, but tuning the annealing conditions is insufficient to resolve this issue. In this study, we developed a novel method to produce accurate nucleotide length PIC at an industrial scale. The length extension was significantly suppressed by the assembly of multiple short polyinosinic acid molecules with one long polycytidylic acid molecule. A newly developed PIC, uPIC100-400, demonstrated a reproducible length and better storage stability than that of corresponding evenly structured PIC. Human dsRNA receptors exhibited equivalent responsiveness to uPIC100-400 and the evenly structured PIC with the same length.

Unraveling the 4n - 1 rule for DNA i-motif stability: base pairs vs. loop lengths.

Previously our laboratory identified that poly-2'-deoxycytidine (dCn) strands of DNA with lengths greater than 12 nucleotides could adopt i-motif folds, while the pH-dependent stabilities follow a 4n - 1 repeat pattern with respect to chain length (J. Am. Chem. Soc., 2017, 139, 4682-4689). Herein, model i-motif folds in which loop configurations were forced by judiciously mutating dC to non-dC nucleotides allowed a structural model to be proposed to address this phenomenon. The model was developed by systematically studying two i-motifs with either an even or odd number of d(C·C)+ hemiprotonated base pairs in the core. First, a trend in the pH-dependent stability vs. loop nucleotide identity was observed: dC > dT ∼ dU ≫ dA ∼ dG. Next, loops comprised of dT nucleotides in the two different core base pair configurations were studied while systematically changing the loop lengths. We found that an i-motif with an even number of base pairs in the core with a single nucleotide in each of the three loops was the most stable, as well as an i-motif with an odd number of core base pairs having one nucleotide in the two exterior loops and three nucleotides in the central loop. A systematic increase in the central loop from 1-4 nucleotides for an odd number of base pairs in the i-motif core reproduced the 4n - 1 repeat pattern observed in the poly-dCn strands. Additional loop configurations were studied to further support the model. The results are discussed with respect to their biological relevance.

Systematic Investigation of Two-Dimensional DNA Nanoassemblies for Construction of a Nonspecific Sensor Array.

We have performed a systematic study to analyze the effect of ssDNA length, nucleobase composition, and the type of two-dimensional nanoparticles (2D-nps) on the desorption response of 36 two-dimensional nanoassemblies (2D-NAs) against several proteins. The studies were performed using fluorescently labeled polyA, polyC, and polyT with 23, 18, 12, and 7 nucleotide-long sequences. The results suggest that the ssDNAs with polyC and longer sequences are more resistant to desorption, compared to their counterparts. In addition, 2D-NAs assembled using WS2 were least susceptible to desorption by the proteins tested, whereas nGO 2D-NAs were the most susceptible nanoassemblies. Later, the results of these systematic studies were used to construct a sensor array for discrimination of seven model proteins (BSA, lipase, alkaline phosphatase, acid phosphatase, protease, ß-galactosidase, and Cytochrome c). Neither the ssDNAs nor the 2D-nps have any specific interaction with the proteins tested. Only the displacement of the ssDNAs from the 2D-np surface was measured upon the disruption of the existing forces within 2D-NAs. A customized sensor array with five 2D-NAs was developed as a result of a careful screening/filtering process. The sensor array was tested against 200 nM of protein targets, and each protein was discriminated successfully. The results suggest that the systematic studies performed using various ssDNAs and 2D-nps enabled the construction of a sensor array without a bind-and-release sensing mechanism. The studies also demonstrate the significance of systematic investigations in the construction of two-dimensional DNA nanoassemblies for functional studies.

Length-Dependent Diblock DNA with Poly-cytosine (Poly-C) as High-Affinity Anchors on Graphene Oxide.

DNA-functionalized graphene oxide (GO) is a popular system for biosensor development and directed materials assembly. Compared to covalent attachment, simple physisorption of DNA has been more popular, and a DNA sequence with a strong affinity on GO is highly desirable. Recently, we found that poly-cytosine (poly-C) DNA can strongly adsorb on many common nanomaterials, including GO. To identify an optimal length of poly-C DNA, we herein designed a series of diblock DNA sequences containing between 0 and 30 cytosines. The displacement of a random sequenced DNA by poly-C DNA was demonstrated, confirming the desired diblock structure on GO with the poly-C block anchoring on the surface and the other block available for hybridization. The adsorption density of poly-C containing DNA did not vary much as the length of the poly-C block increased, suggesting the conformation of the anchoring DNA on the GO was quite independent of the DNA length. With a longer poly-C block, the efficiency of surface hybridization of the other block increased, while nonspecific adsorption of noncomplementary DNA was inhibited more. Compared to poly-adenine (poly-A)-containing DNAs, which were previously used for the same purpose, poly-C DNA adsorption is more stable. Using four types of 15-mer DNA homopolymers as the intended anchoring sequences, the C15 DNA had the best hybridization efficiency. This work has suggested the optimal length for the poly-C block to be 15-mer or longer, and it has provided interesting insights into the DNA/GO biointerface.

Potent Antigen-Adjuvant Delivery System by Conjugation of Mycobacterium tuberculosis Ag85B-HspX Fusion Protein with Arabinogalactan-Poly(I:C) Conjugate.

Protein-based vaccine is promising to improve or replace Mycobacterium bovis BCG vaccine for its specificity, safety, and easy production. However, protein-based vaccine calls for potent adjuvants and improved delivery systems to protect against Mycobacterium tuberculosis. Poly(I:C) is one of the most potent pathogen-associated molecular patterns that signals primarily via TLR3. Arabinogalactan (AG) is a biocompatible polysaccharide that can increase splenocyte proliferation and stimulate macrophages. The AG-poly(I:C) conjugate (AG-P) showed an adjuvant potency through a synergistic interaction of AG and poly(I:C). Ag85B and HspX are two important virulent protein antigens of Mycobacterium tuberculosis and Ag85B-HspX fusion protein (AH) was prepared. An antigen-adjuvant delivery system (AH-AG-P) was developed by conjugation of AH with AG-P to ensure that both AH and AG-P reach the APCs simultaneously. AH-AG-P elicited high AH-specific IgG titers and stimulated lymphocyte proliferation. AH-AG-P provoked the secretion of Th1-type cytokines (TNF-α, IFN-γ, and IL-2) and Th2-type cytokines (IL-4 and IL-10). Pharmacokinetics revealed that conjugation with AG-P could prolong the serum exposure of AH to the immune system. Pharmacodynamics suggested that conjugation with AG-P led to a rapid and intense production of AH-specific IgG. Accordingly, conjugation with AG-P could promote a robust cellular and humoral immune response to AH. Thus, conjugation of AH with a potent adjuvant AG-P is an effective strategy to develop an efficacious protein-based vaccine against Mycobacterium tuberculosis.

Cross-backbone templating; ribodinucleotides made on poly(C).

G(5')pp(5')G synthesis from pG and chemically activated 2MeImpG is accelerated by the addition of complementary poly(C), but affected only slightly by poly(G) and not at all by poly(U) and poly(A). This suggests that 3'-5' poly(C) is a template for uncatalyzed synthesis of 5'-5' GppG, as was poly(U) for AppA synthesis, previously. The reaction occurs at 50 mM mono- and divalent ion concentrations, at moderate temperatures, and near pH 7. The reactive complex at the site of enhanced synthesis of 5'-5' GppG seems to contain a single pG, a single phosphate-activated nucleotide 2 MeImpG, and a single strand of poly(C). Most likely this structure is base-paired, as the poly(C)-enhanced reaction is completely disrupted between 30 and 37 °C, whereas slower, untemplated synthesis of GppG accelerates. More specifically, the reactive center acts as would be expected for short, isolated G nucleotide stacks expanded and ordered by added poly(C). For example, poly(C)-mediated GppG production is very nonlinear in overall nucleotide concentration. Uncatalyzed NppN synthesis is now known for two polymers and their complementary free nucleotides. These data suggest that varied, simple, primordial 3'-5' RNA sequences could express a specific chemical phenotype by encoding synthesis of complementary, reactive, coenzyme-like 5'-5' ribodinucleotides.

Biochemical Characterization of Nonamer Binding Domain of RAG1 Reveals its Thymine Preference with Respect to Length and Position.

RAG complex consisting of RAG1 and RAG2 is a site-specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves recombination signal sequence (RSS), comprising of conserved heptamer and nonamer. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS. To investigate the DNA binding properties of the domain, NBD of murine RAG1 was cloned, expressed and purified. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS or heteroduplex DNA. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G or T were used. Biolayer interferometry studies showed that poly T binding to NBD was robust and comparable to that of 12RSS. More than 23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C or random sequences. Although NBD is indispensable for sequence specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore its activity, suggesting that the overall domain architecture of RAG1 is important. Therefore, we define the sequence requirements of NBD binding to DNA.

Aligned deposition and electrical measurements on single DNA molecules.

A reliable method of deposition of aligned individual dsDNA molecules on mica, silicon, and micro/nanofabricated circuits is presented. Complexes of biotinylated double stranded poly(dG)-poly(dC) DNA with avidin were prepared and deposited on mica and silicon surfaces in the absence of Mg(2+) ions. Due to its positive charge, the avidin attached to one end of the DNA anchors the complex to negatively charged substrates. Subsequent drying with a directional gas flow yields DNA molecules perfectly aligned on the surface. In the avidin-DNA complex only the avidin moiety is strongly and irreversibly bound to the surface, while the DNA counterpart interacts with the substrates much more weakly and can be lifted from the surface and realigned in any direction. Using this technique, avidin-DNA complexes were deposited across platinum electrodes on a silicon substrate. Electrical measurements on the deposited DNA molecules revealed linear IV-characteristics and exponential dependence on relative humidity.

Delivery of cell-penetrating peptide-peptide nucleic acid conjugates by assembly on an oligonucleotide scaffold.

Delivery to intracellular target sites is still one of the main obstacles in the development of peptide nucleic acids (PNAs) as antisense-antigene therapeutics. Here, we designed a self-assembled oligonucleotide scaffold that included a central complementary region for self-assembly and lateral regions complementing the PNAs. Assembly of cell-penetrating peptide (CPP)-PNAs on the scaffold significantly promoted endocytosis of PNAs by at least 10-fold in cell cultures, particularly for scaffolds in which the central complementary region was assembled by poly(guanine) and poly(cytosine). The antisense activity of CPP-PNAs increased by assembly on the scaffold and was further enhanced after co-assembly with endosomolytic peptide (EP)-PNA. This synergistic effect was also observed following the assembly of antigene CPP-PNAs\EP-PNAs on the scaffold. However, antigene activity was only observed by targeting episomal viral DNA or transfected plasmids, but not the chromosome in the cell cultures. In conclusion, assembly on oligonucleotide scaffolds significantly enhanced the antisense-antigene activity of PNAs by promoting endocytosis and endosomal escape. This oligonucleotide scaffold provided a simple strategy for assembly of multiple functional peptide-PNA conjugates, expanding the applications of PNAs and demonstrating the potential of PNAs as antiviral therapeutics.

Nucleobase Recognition by Truncated α-Hemolysin Pores.

The α-hemolysin (αHL) protein nanopore has been investigated previously as a base detector for the strand sequencing of DNA and RNA. Recent findings have suggested that shorter pores might provide improved base discrimination. New work has also shown that truncated-barrel mutants (TBM) of αHL form functional pores in lipid bilayers. Therefore, we tested TBM pores for the ability to recognize bases in DNA strands immobilized within them. In the case of TBMΔ6, in which the barrel is shortened by ∼16 Å, one of the three recognition sites found in the wild-type pore, R1, was almost eliminated. With further mutagenesis (Met113 → Gly), R1 was completely removed, demonstrating that TBM pores can mediate sharpened recognition. Remarkably, a second mutant of TBMΔ6 (Met113 → Phe) was able to bind the positively charged ß-cyclodextrin, am7ßCD, unusually tightly, permitting the continuous recognition of individual nucleoside monophosphates, which would be required for exonuclease sequencing mediated by nanopore base identification.