Effects of BPQ binding on the nonadiabatic dynamics of excited electrons in poly(dG)-poly(dC) DNA under proton irradiation.

The interaction between DNA and small molecules is important for understanding the mechanisms of DNA-based multifunctional devices and has been extensively studied. However, there are few reports on such interactions in irradiation environments. Here, we investigate the nonadiabatic dynamic behaviors of excited electrons in double-stranded DNA bound to BPQ molecule upon proton irradiation, focusing on the energy deposition and electronic excitation dynamics as protons traverse the DNA along different channels. Our results reveal that the presence of BPQ significantly influences charge migration and DNA damage, with notable differences between CGvdW and CGcovalent adducts. The energy deposition process is highly dependent on charge density, and guanine exhibits higher excitation propensity than cytosine due to its structural characteristics. The BPQ molecule enhances DNA charge migration and promotes damage through secondary electron migration. These findings provide insights into the nonadiabatic dynamics of DNA under ionizing radiation and have implications for designing targeted electrophilic organics to improve radiotherapy efficacy.

Allergen Immunotherapy Enhances Airway Epithelial Antiviral Immunity in Patients with Allergic Asthma (VITAL Study): A Double-Blind Randomized Controlled Trial.

Rationale: Allergic asthma is linked to impaired bronchial epithelial secretion of IFNs, which may be causally linked to the increased risk of viral exacerbations. We have previously shown that allergen immunotherapy (AIT) effectively reduces asthma exacerbations and prevents respiratory infections requiring antibiotics; however, whether AIT alters antiviral immunity is still unknown. Objectives: To investigate the effect of house dust mite sublingual AIT (HDM-SLIT) on bronchial epithelial antiviral and inflammatory responses in patients with allergic asthma. Methods: In this double-blind, randomized controlled trial (VITAL [The Effect of Allergen Immunotherapy on Anti-viral Immunity in Patients with Allergic Asthma]), adult patients with HDM allergic asthma received HDM-SLIT 12-SQ or placebo for 24 weeks. Bronchoscopy was performed at baseline and at Week 24, which included sampling for human bronchial epithelial cells. Human bronchial epithelial cells were cultured at baseline and at Week 24 and stimulated with the viral mimic polyinosinic:polycytidylic acid (poly(I:C)). mRNA expression was quantified using qRT-PCR, and protein concentrations were measured using multiplex ELISA. Measurements and Main Results: Thirty-nine patients were randomized to HDM-SLIT (n = 20) or placebo (n = 19). HDM-SLIT resulted in increased polyinosinic:polycytidylic acid-induced expression of IFN-ß at both the gene (P = 0.009) and protein (P = 0.02) levels. IFN-λ gene expression was also increased (P = 0.03), whereas IL-33 tended to be decreased (P = 0.09). On the other hand, proinflammatory cytokines IL-6 (P = 0.009) and TNF-α (tumor necrosis factor-α) (P = 0.08) increased compared with baseline in the HDM-SLIT group. There were no significant changes in TSLP (thymic stromal lymphopoietin), IL-4, IL-13, and IL-10. Conclusions: HDM-SLIT improves bronchial epithelial antiviral resistance to viral infection. These results potentially explain the efficacy of HDM-SLIT in reducing exacerbations in allergic asthma. Clinical trial registered with www.clinicaltrials.gov (NCT04100902).

The long-lasting enigma of polycytidine (polyC) tract.

Long polycytidine (polyC) tracts varying in length from 50 to 400 nucleotides were first described in the 5'-noncoding region (NCR) of genomes of picornaviruses belonging to the Cardio- and Aphthovirus genera over 50 years ago, but the molecular basis of their function is still unknown. Truncation or complete deletion of the polyC tracts in picornaviruses compromises virulence and pathogenicity but do not affect replicative fitness in vitro, suggesting a role as "viral security" RNA element. The evidence available suggests that the presence of a long polyC tract is required for replication in immune cells, which impacts viral distribution and targeting, and, consequently, pathogenic progression. Viral attenuation achieved by reduction of the polyC tract length has been successfully used for vaccine strategies. Further elucidation of the role of the polyC tract in viral replication cycle and its connection with replication in immune cells has the potential to expand the arsenal of tools in the fight against cancer in oncolytic virotherapy (OV). Here, we review the published data on the biological significance and mechanisms of action of the polyC tract in viral pathogenesis in Cardio- and Aphthoviruses.

Peripheral immune challenges elicit differential up-regulation of hippocampal cytokine and chemokine mRNA expression in a mouse model of the 15q13.3 microdeletion syndrome.

The human heterozygous 15q13.3 microdeletion is associated with neuropathological disorders, most prominently with epilepsy and intellectual disability. The 1.5 Mb deletion encompasses six genes (FAN1 [MTMR15], MTMR10, TRPM1, KLF13, OTUD7A, and CHRNA7); all but one (TRPM1) are expressed in the brain. The 15q13.3 microdeletion causes highly variable neurological symptoms, and confounding factors may contribute to a more severe phenotype. CHRNA7 and KLF13 are involved in immune system regulation and altered immune responses may contribute to neurological deficits. We used the Df[h15q13]/+ transgenic mouse model with a heterozygous deletion of the orthologous region (Het) to test the hypothesis that the microdeletion increases innate immune responses compared to wild type (WT). Male and female mice were acutely challenged with the bacteriomimetic lipopolysaccharide (LPS, 0.1 mg/kg, i.p.) or the viral mimetic polyinosinic:polycytidylic acid (Poly(I:C), 5 mg/kg). Hippocampal mRNA expression of pro-inflammatory cytokines and chemokines were determined three hours after injection using quantitative PCR analysis. In controls, expression was not affected by sex or genotype. LPS and Poly(I:C) resulted in significantly increased hippocampal expression of cytokines, chemokines, and interferon-γ (IFNγ), with more robust increases for TNF-α, IL-6, IL-1ß, CXCL1, and CCL2 by LPS, higher induction of IFNγ by Poly(I:C), and similar increases of CCL4 and CCL5 by both agents. Generally, Hets exhibited stronger responses than WT mice, and significant effects of genotype or genotype × treatment interactions were detected for CXCL1 and CCL5, and IL-6, IL-1ß, and CCL4, respectively, after LPS. Sex differences were detected for some targets. LPS but not Poly(I:C), reduced overnight burrowing independent of sex or genotype, suggesting that LPS induced sickness behavior. Thus, mice carrying the microdeletion have an increased innate immune response following a LPS challenge, but further studies will have to determine the extent and mechanisms of altered immune activation and subsequent contributions to 15q13.3 microdeletion associated deficits.

Molecular cloning, expression analysis of interleukin 17D (cysteine knot cytokine) from Amphiprion clarkii and their functional characterization and NFκB pathway activation using FHM cells.

Interleukin 17D (IL-17D), a pro-inflammatory cytokine, is a signature cytokine of T helper 17 (Th17) cells. However, studies characterizing the functions of IL-17D in teleost are scarce. Therefore, we aimed to characterize the properties of IL-17D in Amphiprion clarkii. We performed spatial and temporal expression, AcIL-17D-mediated antibacterial and inflammatory gene expression, NFκB pathway-related gene expression analyses, and bacterial colony counting and cell protection assays. We found that AcIL-17D contains a 630 bp coding sequence and encodes 210 amino acids. The spatial expression analysis of AcIL-17D in 12 tissues showed ubiquitous expression, with the highest expression in the brain, followed by blood and skin. Temporal expression analysis of AcIL-17D in blood showed upregulated expression at 6 and 24 h (polyinosinic: polycytidylic acid and lipopolysaccharide), 12 h (all stimulants), and 48 h (polyinosinic: polycytidylic acid and Vibrio harveyi). AcIL-17D expression in the blood gradually decreased at later hours in response to all the stimulants. After treatment of fathead minnow (FHM) cells with different recombinant AcIL-17D concentrations, the downstream gene expression analysis showed increased expression of antimicrobial genes in the FHM cells, namely [NK-Lysin (NKL), Hepcidin antimicrobial peptide-1 (HAMP-1), Defensin-ß (DEFB1)] and some inflammatory genes such as IL-1ß, TNF-α, IL-11, and STAT3. Further nuclear factor κB (NFκB) subunits (NFκB1, NFκB2, RelA, and Rel-B) showed upregulated gene expression at 12 and 24 h. The bacterial colony counting assay using FHM cells showed lower bacterial colony counts in rAcIL-17D-treated cells than in control. Furthermore, the Water-Soluble Tetrazolium Salt (WST -1) assay confirmed the ability of rAcIL-17D in the protection of FHM cells from bacterial infection and conducted the Hoechst 33342 staining upon treatment with rAcIL-17D and rMBP. Therefore, our findings provide important insights into the activation of IL-17D pathway genes in FHM cells, the protective role of AcIL-17D against bacterial infection, and host defense mechanisms in teleost.

Unique Features of the Immune Response in BTBR Mice.

Inflammation plays a considerable role in the pathogenesis of many diseases, including neurodegenerative and psychiatric ones. Elucidation of the specific features of an immune response in various model organisms, and studying the relation of these features with the behavioral phenotype, can improve the understanding of the molecular mechanisms of many psychopathologies. In this work, we focused on BTBR mice, which have a pronounced autism-like behavioral phenotype, elevated levels of oxidative-stress markers, an abnormal immune response, several structural aberrations in the brain, and other unique traits. Although some studies have already shown an abnormal immune response in BTBR mice, the existing literature data are still fragmentary. Here, we used inflammation induced by low-dose lipopolysaccharide, polyinosinic:polycytidylic acid, or their combinations, in mice of strains BTBR T+Itpr3tf/J (BTBR) and C57BL6/J. Peripheral inflammation was assessed by means of a complete blood count, lymphocyte immunophenotyping, and expression levels of cytokines in the spleen. Neuroinflammation was evaluated in the hypothalamus and prefrontal cortex by analysis of mRNA levels of proinflammatory cytokines (tumor necrosis factor, Tnf), (interleukin-1 beta, Il-1ß), and (interleukin-6, Il-6) and of markers of microglia activation (allograft inflammatory factor 1, Aif1) and astroglia activation (glial fibrillary acidic protein, Gfap). We found that in both strains of mice, the most severe inflammatory response was caused by the administration of polyinosinic:polycytidylic acid, whereas the combined administration of the two toll-like receptor (TLR) agonists did not enhance this response. Nonetheless, BTBR mice showed a more pronounced response to low-dose lipopolysaccharide, an altered lymphocytosis ratio due to an increase in the number of CD4+ lymphocytes, and high expression of markers of activated microglia (Aif1) and astroglia (Gfap) in various brain regions as compared to C57BL6/J mice. Thus, in addition to research into mechanisms of autism-like behavior, BTBR mice can be used as a model of TLR3/TLR4-induced neuroinflammation and a unique model for finding and evaluating the effectiveness of various TLR antagonists aimed at reducing neuroinflammation.

A cytosine-rich splice regulatory determinant enforces functional processing of the human α-globin gene transcript.

The establishment of efficient and stable splicing patterns in terminally differentiated cells is critical to maintenance of specific functions throughout the lifespan of an organism. The human α-globin (hα-globin) gene contains 3 exons separated by 2 short introns. Naturally occurring α-thalassemia mutations that trigger aberrant splicing have revealed the presence of cryptic splice sites within the hα-globin gene transcript. How cognate (functional) splice sites are selectively used in lieu of these cryptic sites has remained unexplored. Here we demonstrate that the preferential selection of a cognate splice donor essential to functional splicing of the hα-globin transcript is dependent on the actions of an intronic cytosine (C)-rich splice regulatory determinant and its interacting polyC-binding proteins. Inactivation of this determinant by mutation of the C-rich element or by depletion of polyC-binding proteins triggers a dramatic shift in splice donor activity to an upstream, out-of-frame, cryptic donor. The essential role of the C-rich element in hα-globin gene expression is supported by its coevolution with the cryptic donor site in primate species. These data lead us to conclude that an intronic C-rich determinant enforces functional splicing of the hα-globin transcript, thus acting as an obligate determinant of hα-globin gene expression.

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.

Enterovirus 71 antagonizes the inhibition of the host intrinsic antiviral factor A3G.

Although the host restriction factor APOBEC3G (A3G) has broad spectrum antiviral activity, whether A3G inhibits enterovirus 71 (EV71) has been unclear until now. In this study, we demonstrated for the first time that A3G could inhibit EV71 virus replication. Silencing A3G in H9 cells enhanced EV71 replication, and EV71 replication was lower in H9 cells expressing A3G than in Jurkat cells without A3G expression, indicating that the EV71 inhibition was A3G-specific. Further investigation revealed that A3G inhibited the 5'UTR activity of EV71 by competitively binding to the 5'UTR through its nucleic acid binding activity. This binding impaired the interaction between the 5'UTR and the host protein poly(C)-binding protein 1 (PCBP1), which is required for the synthesis of EV71 viral proteins and RNA. On the other hand, we found that EV71 overcame A3G suppression through its non-structural protein 2C, which induced A3G degradation through the autophagy-lysosome pathway. Our research provides new insights into the interplay mechanisms of A3G and single-stranded positive RNA viruses.

Capping-RACE: a simple, accurate, and sensitive 5' RACE method for use in prokaryotes.

Rapid amplification of cDNA ends (RACE) is a prevalent technique used to obtain the 5' ends of transcripts. Several different 5' RACE methods have been developed, and one particularly simple and efficient approach called CapFinder relies on the 5' cap-dependent template-switching that occurs in eukaryotes. However, most prokaryotic transcripts lack a 5' cap structure. Here, we report a procedure to capture primary transcripts based on capping the 5' triphosphorylated RNA in prokaryotes. Primary transcripts were first treated with vaccinia capping enzyme to add a 5' cap structure. First-strand cDNA was then synthesized using Moloney murine leukaemia virus reverse transcriptase. Finally, a template-switching oligonucleotide with a tail containing three ribonucleic acid guanines was hybridized to the cDNA 3' poly(C) and further used as template for reverse transcriptase. It is oligonucleotide sequence independent and is more sensitive compared to RLM-RACE. This approach specifically identified the transcription start sites of ompA, sodB and shiA in Escherichia coli and of ompA, rne and rppH in Brucella melitensis. Furthermore, we also successfully identified the transcription start sites of small noncoding genes ryhB and micC in E. coli and bsnc135 and bsnc149 in B. melitensis. Our findings suggest that Capping-RACE is a simple, accurate, and sensitive 5' RACE method for use in prokaryotes.

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.

How iron is handled in the course of heme catabolism: Integration of heme oxygenase with intracellular iron transport mechanisms mediated by poly (rC)-binding protein-2.

Heme and iron are essential to almost all forms of life. The strict maintenance of heme and iron homeostasis is essential to prevent cellular toxicity and the existence of systemic and intracellular regulation is fundamental. Cytosolic heme can be catabolized and detoxified by heme oxygenases (HOs). Interestingly, free heme detoxification through HOs results in the production of free ferrous iron, which can be potentially hazardous for cells. Recently, the intracellular iron chaperone, poly (rC)-binding protein 2 (PCBP2), has been identified, which can be involved in accepting iron after heme catabolism as well as intracellular iron transport. In fact, HO1, NADPH-cytochrome P450 reductase, and PCBP2 form a functional unit that integrates the catabolism of heme with the binding and transport of iron by PCBP2. In this review, we provide an overview of our understanding of the iron chaperones and discuss the mechanism how iron chaperones bind iron released during the process of heme degradation.

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.

Polycytidylation of mitochondrial mRNAs in Chlamydomonas reinhardtii.

The unicellular photosynthetic organism, Chlamydomonas reinhardtii, represents a powerful model to study mitochondrial gene expression. Here, we show that the 5'- and 3'-extremities of the eight Chlamydomonas mitochondrial mRNAs present two unusual characteristics. First, all mRNAs start primarily at the AUG initiation codon of the coding sequence which is often marked by a cluster of small RNAs. Second, unusual tails are added post-transcriptionally at the 3'-extremity of all mRNAs. The nucleotide composition of the tails is distinct from that described in any other systems and can be partitioned between A/U-rich tails, predominantly composed of Adenosine and Uridine, and C-rich tails composed mostly of Cytidine. Based on 3' RACE experiments, 22% of mRNAs present C-rich tails, some of them composed of up to 20 consecutive Cs. Polycytidylation is specific to mitochondria and occurs primarily on mRNAs. This unprecedented post-transcriptional modification seems to be a specific feature of the Chlorophyceae class of green algae and points out the existence of novel strategies in mitochondrial gene expression.

Global characterization of the oocyte-to-embryo transition in Caenorhabditis elegans uncovers a novel mRNA clearance mechanism.

The oocyte-to-embryo transition (OET) is thought to be mainly driven by post-transcriptional gene regulation. However, expression of both RNAs and proteins during the OET has not been comprehensively assayed. Furthermore, specific molecular mechanisms that regulate gene expression during OET are largely unknown. Here, we quantify and analyze transcriptome-wide, expression of mRNAs and thousands of proteins in Caenorhabditis elegans oocytes, 1-cell, and 2-cell embryos. This represents a first comprehensive gene expression atlas during the OET in animals. We discovered a first wave of degradation in which thousands of mRNAs are cleared shortly after fertilization. Sequence analysis revealed a statistically highly significant presence of a polyC motif in the 3' untranslated regions of most of these degraded mRNAs. Transgenic reporter assays demonstrated that this polyC motif is required and sufficient for mRNA degradation after fertilization. We show that orthologs of human polyC-binding protein specifically bind this motif. Our data suggest a mechanism in which the polyC motif and binding partners direct degradation of maternal mRNAs. Our data also indicate that endogenous siRNAs but not miRNAs promote mRNA clearance during the OET.

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.