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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Application of OmpF nanochannel forming protein in polynucleotide sequence recognition.

Recognition of the sequence of human genome sequence is vital to address malfunctions occurring at molecular, cellular and tissue levels and requires a great deal of time, cost and efforts. Thus, various synthetic and natural pores were considered to fabricate high-throughput systems capable to fulfill the task in an efficient manner. Here, voltage gating OmpF nanochannel, whose structure is known at an atomic level, was used to recognize and differentiate between polynucleotide primers through voltage clamp technique. Our results showed that poly(T) occasionally blocked the channel at both polarities, while poly(C) and poly(G) obstructed it only at positive polarity. The channel was blocked at potential differences of as low as 80 mV in the presence of poly(T). The conductance of channel decreased in the presence of poly(C) and poly(G) by 61 and 5% respectively. Analysis of the number of events showed that poly(T) caused more closing events at higher voltages, while poly(G) and poly(C) induced it at lower voltages. Application of the hazard function as a statistical parameter and analysis of event closing times in various voltages demonstrated the most efficient differentiation at 60 mV. The results of practical and theoretical approaches presented here show that OmpF porin channel possesses the structural and dynamic characteristics required to be considered as a biosensor capable for continuous polynucleotide sequencing.

Phase variable genes of Campylobacter jejuni exhibit high mutation rates and specific mutational patterns but mutability is not the major determinant of population structure during host colonization.

Phase variation of surface structures occurs in diverse bacterial species due to stochastic, high frequency, reversible mutations. Multiple genes of Campylobacter jejuni are subject to phase variable gene expression due to mutations in polyC/G tracts. A modal length of nine repeats was detected for polyC/G tracts within C. jejuni genomes. Switching rates for these tracts were measured using chromosomally-located reporter constructs and high rates were observed for cj1139 (G8) and cj0031 (G9). Alteration of the cj1139 tract from G8 to G11 increased mutability 10-fold and changed the mutational pattern from predominantly insertions to mainly deletions. Using a multiplex PCR, major changes were detected in 'on/off' status for some phase variable genes during passage of C. jejuni in chickens. Utilization of observed switching rates in a stochastic, theoretical model of phase variation demonstrated links between mutability and genetic diversity but could not replicate observed population diversity. We propose that modal repeat numbers have evolved in C. jejuni genomes due to molecular drivers associated with the mutational patterns of these polyC/G repeats, rather than by selection for particular switching rates, and that factors other than mutational drift are responsible for generating genetic diversity during host colonization by this bacterial pathogen.

[Degradation of polyribonucleotides: biocatalysis and the monitoring of products].

Macroporous monolithic material containing covalently linked ribonuclease A was used to create high-performance flow heterogeneous biocatalysts (bioreactors). The kinetic parameters of the degradation of polycytidylic acid were identified, and the properties of the obtained systems were compared. A HPLC method has been developed for monitoring products of biocatalytic degradation of RNA, and the possibility of using biocatalytic and HPLC columns in RNA degradation processes in a multicomponent mixture of biological molecules was shown.

Oligopyrenotides: chiral nanoscale templates for chromophore assembly.

Getting organized: DNA-like supramolecular polymers formed of short oligopyrenotides serve as a helical scaffold for the molecular assembly of ligands. The cationic porphyrin meso-tetrakis(1-methylpyridin-4-yl)porphyrin interacts with the helical polymers in a similar way as with poly(dA:dT).

Discovery of the DNA "genetic code" for abiological gold nanoparticle morphologies.

DNA is in control: Different combinations of DNA nucleotides can control the shape and surface roughness of gold nanoparticles during their synthesis. These nanoparticles were synthesized in the presence of either homogenous oligonucleotides or mixed-base oligonucleotides using gold nanoprisms as seeds. The effect of the individual DNA bases and their combinations on shape control are shown in the figure.

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.

NaDDBS as a dispersion agent for multiwalled carbon nanotubes in capillary EKC separation of nucleotides.

A novel pseudostationary phase (PSP) of multiwalled carbon nanotubes (MWCNTs) dispersed with sodium dodecylbenzenesulfonate (NaDDBS) was used for the EKC separation of nucleotides. NaDDBS has a long hydrophobic chain and a benzylsulfonate group. It suspends more MWCNTs (about 100-fold) than SDS, and the π-π interaction between the benzene ring of NaDDBS and MWCNTs prolongs the slurry suspension time. Using NaDDBS as a surfactant can reduce the required amount of MWCNTs and decrease the baseline noise. To produce a stable suspension, the optimum ratio (w/w) of MWCNTs to NaDDBS was investigated with turbidimetry. In this context, several parameters affecting EKC separation were studied, including buffer pH, composition, concentration, and the organic modifier. Use of NaDDBS (8 mg/L)/MWCNTs (0.8 mg/L) as the PSP in a phosphate buffer (30 mM, pH 8) yielded complete resolution of seven geometric isomers of a nucleoside monophosphate. In stacking mode, with 10% MeOH in the sample plug, the mixture of nucleoside mono-, di-, and tri-phosphates was satisfactorily separated in phosphate buffer (50 mM, pH 9). The results indicate that nucleotides with bases containing more electron-withdrawing groups interact more strongly with MWCNTs. The system has been used to separate oligonucleotides, and to analyze nucleotides in a complex matrix sample.

Long-lived fluorescence of homopolymeric guanine-cytosine DNA duplexes.

The fluorescence spectrum of the homopolymeric double helix poly(dG) x poly(dC) is dominated by emission decaying on the nanosecond time-scale, as previously reported for the alternating homologue poly(dGdC) x poly(dGdC). Thus, energy trapping over long periods of time is a common feature of GC duplexes which contrast with AT duplexes. The impact of such behaviour on DNA photodamage needs to be evaluated.