Partner Strand Research Articles

Fast and precise multi-site mutagenesis on linear DNA fragments

The effectiveness and scalability of site-directed mutagenesis are constrained by the limited number of mutations and the intricate cloning process required for isolation of the target sequence. Here, we present a method for precise introduction of multiple non-contiguous mutations. The mutant strands are collected through one specially designed magnetic bead separation in alkaline conditions, efficiently removing their complementary partner strands with the original sequences. In a proof-of-concept test, a green fluorescent protein (GFP) was simultaneously mutated in 1–3 specific amino acids, successfully shifting its fluorescence spectrum. The precise mutation rates for single-, double- and triple-site mutations reached 100%, 76% and 70%, respectively. This multiple non-contiguous mutagenesis method may offer a fast and cost-effective approach for customizable construction of gene library.

MiR-155-3p: processing by-product or rising star in immunity and cancer?

MicroRNAs (miRNAs) are key players in gene regulation that target specific mRNAs for degradation or translational repression. Each miRNA is synthesized as a miRNA duplex comprising two strands (5p and 3p). However, only one of the two strands becomes active and is selectively incorporated into the RNA-induced silencing complex in a process known as miRNA strand selection. Recently, significant progress has been made in understanding the factors and processes involved in strand selection. Here, we explore the selection and functionality of the miRNA star strand (either 5p or 3p), which is generally present in the cell at low levels compared to its partner strand and, historically, has been thought to possess no biological activity. We also highlight the concepts of miRNA arm switching and miRNA isomerism. Finally, we offer insights into the impact of aberrant strand selection on immunity and cancer. Leading us through this journey is miR-155, a well-established regulator of immunity and cancer, and the increasing evidence that its 3p strand plays a role in these arenas. Interestingly, the miR-155-5p/-3p ratio appears to vary dependent on the timing of the immune response, and the 3p strand seems to play a regulatory role upon its partner 5p strand.

A pH and Mg2+-Responsive Molecular Switch Based on a Stable DNA Minidumbbell Bearing 5' and 3'-Overhangs.

Minidumbbell (MDB) is a non-B DNA structure of which the thermodynamic stability is sensitive to a chemical environment such as pH, serving as a potential structural motif in constructing DNA-based molecular switches. This work aims to design thermodynamically stable MDB structures bearing 5′ and 3′-overhanging deoxyribonucleotides in order to examine the possibility of MDB to be functionalized. Via making use of 5-methylcytosine and adjusting the pH of solution to be acidic, MDBs bearing 1-nucleotide (nt) or 2-nt overhanging residues at the 5′ and 3′-ends have been obtained. Based on one of the new MDB sequences, we have designed a molecular switch that could respond to dual inputs of pH and Mg2+. The MDB strand and its partner strand formed a duplex (the “ON” state) upon inputting pH 7 and Mg2+, whereas the duplex dissociated to restore the MDB structure (the “OFF” state) upon inputting pH 5 and EDTA. The demonstration on the ability of MDB to sustain 5′ and 3′-overhanging residues and the construction of a pH and Mg2+-responsive molecular switch will extend the application of MDB structures in dynamic DNA nanotechnology.

Elucidation of proteostasis defects caused by osteogenesis imperfecta mutations in the collagen-α2(I) C-propeptide domain

Intracellular collagen assembly begins with the oxidative folding of ∼30-kDa C-terminal propeptide (C-Pro) domains. Folded C-Pro domains then template the formation of triple helices between appropriate partner strands. Numerous C-Pro missense variants that disrupt or delay triple-helix formation are known to cause disease, but our understanding of the specific proteostasis defects introduced by these variants remains immature. Moreover, it is unclear whether or not recognition and quality control of misfolded C-Pro domains is mediated by recognizing stalled assembly of triple-helical domains or by direct engagement of the C-Pro itself. Here, we integrate biochemical and cellular approaches to illuminate the proteostasis defects associated with osteogenesis imperfecta-causing mutations within the collagen-α2(I) C-Pro domain. We first show that "C-Pro-only" constructs recapitulate key aspects of the behavior of full-length Colα2(I) constructs. Of the variants studied, perhaps the most severe assembly defects are associated with C1163R C-Proα2(I), which is incapable of forming stable trimers and is retained within cells. We find that the presence or absence of an unassembled triple-helical domain is not the key feature driving cellular retention versus secretion. Rather, the proteostasis network directly engages the misfolded C-Pro domain itself to prevent secretion and initiate clearance. Using MS-based proteomics, we elucidate how the endoplasmic reticulum (ER) proteostasis network differentially engages misfolded C1163R C-Proα2(I) and targets it for ER-associated degradation. These results provide insights into collagen folding and quality control with the potential to inform the design of proteostasis network-targeted strategies for managing collagenopathies.

Transient up-regulation of miR-155-3p by lipopolysaccharide in primary human monocyte-derived macrophages results in RISC incorporation but does not alter TNF expression.

Background: The innate immune response is a tightly regulated process that reacts rapidly in response to pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). Evidence is accumulating that microRNAs contribute to this, although few studies have examined the early events that constitute the "primary" response. Methods: LPS-dependent changes to miRNA expression were studied in primary human monocyte-derived macrophages (1°MDMs). An unbiased screen by microarray was validated by qPCR and a method for the absolute quantitation of miRNAs was also developed, utilising 5' phosphorylated RNA oligonucleotide templates. RNA immunoprecipitation was performed to explore incorporation of miRNAs into the RNA-induced silencing complex (RISC). The effect of miRNA functional inhibition on TNF expression (mRNA and secretion) was investigated. Results: Of the 197 miRNAs expressed in 1°MDMs, only five were induced >1.5-fold. The most strongly induced was miR-155-3p, the partner strand to miR-155-5p, which are both derived from the MIR155HG/BIC gene (pri-miR-155). The abundance of miR-155-3p was induced transiently ~250-fold at 2-4hrs and then returned towards baseline, mirroring pri-miR-155. Other PAMPs, IL-1β, and TNF caused similar responses. IL-10, NF-κB, and JNK inhibition reduced these responses, unlikecytokine-suppressing mycolactone. Absolute quantitation revealed that miRNA abundance varies widely from donor-to-donor, and showed that miR-155-3p abundance is substantially less than miR-155-5p in unstimulated cells. However, at its peak there were 446-1,113 copies/cell, and miR-155-3p was incorporated into the RISC with an efficiency similar to miR-16-5p and miR-155-5p. Inhibition of neither miRNA affected TNF secretion after 2hrs in 1°MDMs, but technical challenges here are noted. Conclusions: Dynamic regulation of miRNAs during the primary response is rare, with the exception of miR-155-3p. Further work is required to establish whether its low abundance, even at the transient peak, is sufficient for biological activity and to determine whether there are specific mechanisms determining its biogenesis from miR-155 precursors.

Transient up-regulation of miR-155-3p by lipopolysaccharide in primary human monocyte-derived macrophages results in RISC incorporation but does not alter TNF expression

Background: The innate immune response is a tightly regulated process that reacts rapidly in response to pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). Evidence is accumulating that microRNAs contribute to this, although few studies have examined the early events that constitute the “primary” response. Methods: LPS-dependent changes to miRNA expression were studied in primary human monocyte-derived macrophages (1°MDMs). An unbiased screen by microarray was validated by qPCR and a method for the absolute quantitation of miRNAs was also developed, utilising 5’ phosphorylated RNA oligonucleotide templates. RNA immunoprecipitation was performed to explore incorporation of miRNAs into the RNA-induced silencing complex (RISC). The effect of miRNA functional inhibition on TNF expression (mRNA and secretion) was investigated. Results: Of the 197 miRNAs expressed in 1°MDMs, only five were induced >1.5-fold. The most strongly induced was miR-155-3p, the partner strand to miR-155-5p, which are both derived from the MIR155HG/BIC gene (pri-miR-155). The abundance of miR-155-3p was induced transiently ~250-fold at 2-4hrs and then returned towards baseline, mirroring pri-miR-155. Other PAMPs, IL-1β, and TNF caused similar responses. IL-10, NF-κB, and JNK inhibition reduced these responses, unlike cytokine-suppressing mycolactone. Absolute quantitation revealed that miRNA abundance varies widely from donor-to-donor, and showed that miR-155-3p abundance is substantially less than miR-155-5p in unstimulated cells. However, at its peak there were 446-1,113 copies/cell, and miR-155-3p was incorporated into the RISC with an efficiency similar to miR-16-5p and miR-155-5p. Inhibition of neither miRNA affected TNF secretion after 2hrs in 1°MDMs, but technical challenges here are noted. Conclusions: Dynamic regulation of miRNAs during the primary response is rare, with the exception of miR-155-3p. Further work is required to establish whether its low abundance, even at the transient peak, is sufficient for biological activity and to determine whether there are specific mechanisms determining its biogenesis from miR-155 precursors

Retraction: The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA.

When a cell divides to form a new cell, it must also copy its DNA. An important step for starting DNA replication is to break or “melt” the bonds between the two strands of DNA that make up the double helix. This happens at a specific site on the DNA called the replication origin, and allows double-stranded DNA to partially unwind into two single strands. Each strand acts as a template to form a new copy of its partner strand. In many organisms, ring-shaped proteins called helicases attach directly to double-stranded DNA to melt the bonds at the replication origin. Viruses also have helicases that they can use to hijack the cell’s replication machinery and get it to copy the viral DNA. One such helicase called Large T antigen is an important part of a tumor virus called simian virus 40. Large T has a ‘hexameric’ structure, similar to the cell’s own helicases, and researchers often use it as a model for studying the replication process. However, key questions remain. How do hexameric helicases interact with the double-stranded DNA replication origin? And what are the molecular mechanisms by which these helicases melt bonds in double-stranded DNA? Gai et al. used a technique called X-ray crystallography to examine the molecular structure of the Large T helicase when it is attached to DNA. The resulting structures show that six subunits of Large T helicase assemble a ring around the replication origin, each making unique bonds with the DNA. The helicase ring squeezes the DNA, partially breaking the bonds between the DNA strands and causing local melting of the DNA. With the understanding of how the Large T hexamer helicase interacts with its DNA replication origin and how its assembly initiates DNA melting, the future challenge is to explain the process by which the partially melted origin opens up fully for replication to begin.

Transient up-regulation of miR-155-3p by lipopolysaccharide in primary human monocyte-derived macrophages results in RISC incorporation but does not alter TNF expression

Background: The innate immune response is a tightly regulated process that reacts rapidly in response to pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS). Evidence is accumulating that microRNAs contribute to this, although few studies have examined the early events that constitute the "primary" response. Methods: LPS-dependent changes to miRNA expression were studied in primary human monocyte-derived macrophages (1°MDMs). An unbiased screen by microarray was validated by qPCR and a method for the absolute quantitation of miRNAs was also developed, utilising 5' phosphorylated RNA oligonucleotide templates. RNA immunoprecipitation was performed to explore incorporation of miRNAs into the RNA-induced silencing complex (RISC). The effect of miRNA functional inhibition on TNF expression (mRNA and secretion) was investigated. Results: Of the 197 miRNAs expressed in 1°MDMs, only five were induced >1.5-fold. The most strongly induced was miR-155-3p, the partner strand to miR-155-5p, which are both derived from the BIC gene (B cell integration cluster, MIR155HG). The abundance of miR-155-3p was induced transiently ~250-fold at 2-4hrs and then returned towards baseline, mirroring the BIC mRNA. Other PAMPs, IL-1β, and TNF caused similar responses. IL-10, NF-κB, and JNK inhibition suppressed these responses, unlikecytokine-suppressing mycolactone. Absolute quantitation showed that miRNA abundance varies widely from donor-to-donor, and showed that miR-155-3p abundance is substantially less than miR-155-5p in unstimulated cells. However, at its peak there were 446-1,113 copies/cell, and miR-155-3p was incorporated into the RISC with an efficiency similar to miR-16-5p and miR-155-5p. Inhibition of neither miRNA affected TNF expression in 1°MDMs, but technical challenges here are noted. Conclusions: Dynamic regulation of miRNAs during the primary response is rare, with the exception of miR-155-3p, which transiently achieves levels that might have a biological effect. Further work on this candidate would need to overcome the technical challenges of the broad-ranging effects of liposomes on 1°MDMs.

Lesion Sensing during Initial Binding by Yeast XPC/Rad4: Toward Predicting Resistance to Nucleotide Excision Repair.

Nucleotide excision repair (NER) excises a variety of environmentally derived DNA lesions. However, NER efficiencies for structurally different DNA lesions can vary by orders of magnitude; yet the origin of this variance is poorly understood. Our goal is to develop computational strategies that predict and identify the most hazardous, repair-resistant lesions from the plethora of such adducts. In the present work, we are focusing on lesion recognition by the xeroderma pigmentosum C protein complex (XPC), the first and required step for the subsequent assembly of factors needed to produce successful NER. We have performed molecular dynamics simulations to characterize the initial binding of Rad4, the yeast orthologue of human XPC, to a library of 10 different lesion-containing DNA duplexes derived from environmental carcinogens. These vary in lesion chemical structures and conformations in duplex DNA and exhibit a wide range of relative NER efficiencies from repair resistant to highly susceptible. We have determined a promising set of structural descriptors that characterize initial binding of Rad4 to lesions that are resistant to NER. Key initial binding requirements for successful recognition are absent in the repair-resistant cases: There is little or no duplex unwinding, very limited interaction between the β-hairpin domain 2 of Rad4 and the minor groove of the lesion-containing duplex, and no conformational capture of a base on the lesion partner strand. By contrast, these key binding features are present to different degrees in NER susceptible lesions and correlate to their relative NER efficiencies. Furthermore, we have gained molecular understanding of Rad4 initial binding as determined by the lesion structures in duplex DNA and how the initial binding relates to the repair efficiencies. The development of a computational strategy for identifying NER-resistant lesions is grounded in this molecular understanding of the lesion recognition mechanism.

Orthogonal γPNA Dimerization Domains Empower DNA Binders with Cooperativity and Versatility Mimicking that of Transcription Factor Pairs.

Synthetic molecules capable of DNA binding and mimicking cooperation of transcription factor (TF) pairs have long been considered a promising tool for manipulating gene expression. Our previously reported Pip-HoGu system, a programmable DNA binder pyrrole-imidazole polyamides (PIPs) conjugated to host-guest moiety, defined a general framework for mimicking cooperative TF pair-DNA interactions. Here, we supplanted the cooperation modules with left-handed (LH) γPNA modules: i.e., PIPs conjugated with nucleic acid-based cooperation system (Pip-NaCo). LH γPNA was chosen because of its bioorthogonality, sequence-specific interaction, and high binding affinity toward the partner strand. From the results of the Pip-NaCo system, cooperativity is highly comparable to the natural TF pair-DNA system, with a minimum energetics of cooperation of -3.27 kcal mol-1 . Moreover, through changing the linker conjugation site, binding mode, and the length of γPNAs sequence, the cooperative energetics of Pip-NaCo can be tuned independently and rationally. The current Pip-NaCo platform might also have the potential for precise manipulation of biological processes through the construction of triple to multiple heterobinding systems.

A miR-29b Byproduct Sequence Exhibits Potent Tumor-Suppressive Activities via Inhibition of NF-κB Signaling in KRAS-Mutant Colon Cancer Cells.

We previously demonstrated that miR-29b-3p is a hopeful miRNA-based therapy against colorectal cancer. In this study, we aimed to clarify a value of miR-29b-1-5p as a next-generation treatment, especially for KRAS-mutant colorectal cancer. RT-PCR assay showed that the expression of miR-29b-3p was high, and its partner strand, miR-29b-1-5p, level was only negligible in clinical colorectal cancer samples. Mimic-miR-29b-1-5p significantly inhibited proliferation of KRAS-mutant colorectal cancer cell lines DLD1 and SW480 and KRAS wild-type HT29 cells. Proliferative activity was further examined by either miR-29b-1-5p strand or its opposite complementary sequence because miR-29b-1-5p is a passenger miRNA and may have no physiologic function. We found that completely opposite complementary strand to miR-29b-1-5p, but not miR-29b-1-5p, possessed a potent antitumor effect and named this byproduct miRNA sequence "MIRTX." MIRTX directly targeted the 3'-UTR of CXCR2 and PIK3R1 mRNA and suppressed the NF-κB signaling pathway in KRAS-mutated colorectal cancer cells. MIRTX induced apoptosis in DLD1 with downregulation of antiapoptotic BCL2, BCL-xL, and MCL1 and upregulation of cleaved caspase-3 and cleaved PARP. In mouse xenograft models, systemic administration of MIRTX using a super carbonate apatite as a delivery vehicle significantly inhibited tumor growth of DLD1 and HT29 cells without any particular toxicities. In conclusion, these findings indicate that inhibition of NF-κB signaling by this novel miRNA-based therapeutic could be a promising treatment against refractory KRAS-mutant colorectal cancer and KRAS wild-type colorectal cancer. Mol Cancer Ther; 17(5); 977-87. ©2018 AACR.

Urea Mimics Nucleobases by Preserving the Helical Integrity of B-DNA Duplexes via Hydrogen Bonding and Stacking Interactions.

Urea lesions are formed in DNA because of free radical damage of the thymine base, and their occurrence in DNA blocks DNA polymerases, which has deleterious consequences. Recently, it has been shown that urea is capable of forming hydrogen bonding and stacking interactions with nucleobases, which are responsible for the unfolding of RNA in aqueous urea. Base pairing and stacking are inherent properties of nucleobases; because urea is able to form both, this study attempts to investigate if urea can mimic nucleobases in the context of nucleic acid structures by examining the effect of introducing urea lesions complementary to the four different nucleobases on the overall helical integrity of B-DNA duplexes and their thermodynamic stabilities using molecular dynamics (MD) simulations. The MD simulations resulted in stable duplexes without significant changes in the global B-DNA conformation. The urea lesions occupy intrahelical positions by forming hydrogen bonds with nitrogenous nucleobases, in agreement with experimental results. Furthermore, these urea lesions form hydrogen bonding and stacking interactions with other nucleobases of the same and partner strands, analogous to nucleobases in typical B-DNA duplexes. Direct hydrogen bond interactions are observed for the urea-purine pairs within DNA duplexes, whereas two different modes of pairing, namely, direct hydrogen bonds and water-mediated hydrogen bonds, are observed for the urea-pyrimidine pairs. The latter explains the complexities involved in interpreting the experimental nuclear magnetic resonance data reported previously. Binding free energy calculations were further performed to confirm the thermodynamic stability of the urea-incorporated DNA duplexes with respect to pure duplexes. This study suggests that urea mimics nucleobases by pairing opposite all four nucleobases and maintains the overall structure of the B-DNA duplexes.

Phosphoramidate Ligation of Oligonucleotides in Nanoscale Structures.

The folding of long DNA strands into designed nanostructures has evolved into an art. Being based on linear chains only, the resulting nanostructures cannot readily be transformed into covalently linked frameworks. Covalently linking strands in the context of folded DNA structures requires a robust method that avoids sterically demanding reagents or enzymes. Here we report chemical ligation of the 3'-amino termini of oligonucleotides and 5'-phosphorylated partner strands in templated reactions that produce phosphoramidate linkages. These reactions produce inter-nucleotide linkages that are isoelectronic and largely isosteric to phosphodiesters. Ligations were performed at three levels of complexity, including the extension of branched DNA hybrids and the ligation of six scaffold strands in a small origami.

Mechanistic insights into the recognition of 5-methylcytosine oxidation derivatives by the SUVH5 SRA domain.

5-Methylcytosine (5 mC) is associated with epigenetic gene silencing in mammals and plants. 5 mC is consecutively oxidized to 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by ten-eleven translocation enzymes. We performed binding and structural studies to investigate the molecular basis of the recognition of the 5 mC oxidation derivatives in the context of a CG sequence by the SET- and RING-associated domain (SRA) of the SUVH5 protein (SUVH5 SRA). Using calorimetric measurements, we demonstrate that the SRA domain binds to the hydroxymethylated CG (5hmCG) DNA duplex in a similar manner to methylated CG (5mCG). Interestingly, the SUVH5 SRA domain exhibits weaker affinity towards carboxylated CG (5caCG) and formylated CG (5fCG). We report the 2.6 Å resolution crystal structure of the SUVH5 SRA domain in a complex with fully hydroxymethyl-CG and demonstrate a dual flip-out mechanism, whereby the symmetrical 5hmCs are simultaneously extruded from the partner strands of the DNA duplex and are positioned within the binding pockets of individual SRA domains. The hydroxyl group of 5hmC establishes both intra- and intermolecular interactions in the binding pocket. Collectively, we show that SUVH5 SRA recognizes 5hmC in a similar manner to 5 mC, but exhibits weaker affinity towards 5 hmC oxidation derivatives.

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO 4 and 5′-OH ends

There are many biological contexts in which DNA damage generates "dirty" breaks with 3'-PO4 (or cyclic-PO4) and 5'-OH ends that cannot be sealed by DNA ligases. Here we show that the Escherichia coli RNA ligase RtcB can splice these dirty DNA ends via a unique chemical mechanism. RtcB transfers GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form a "capped" 3' end structure, DNA3'pp5'G. When a suitable DNA 5'-OH end is available, RtcB catalyzes attack of the 5'-OH on DNA3'pp5'G to form a 3'-5' phosphodiester splice junction. Our findings unveil an enzymatic capacity for DNA 3' capping and the sealing of DNA breaks with 3'-PO4 and 5'-OH termini, with implications for DNA repair and DNA rearrangements.

Altering Colloidal Surface Functionalization Using DNA Encapsulated Inside Monodisperse Gelatin Microsphere Templates

Soluble oligonucleotides are typically introduced to bulk solution to promote hybridization activity on DNA-functionalized surfaces. Here, an alternative approach is explored by encapsulating secondary target strands inside semipermeable colloidal satellite assemblies, then triggering their release at 37 °C for subsequent surface hybridization activity. To prepare DNA-loaded satellite assemblies, uniform gelatin microspheres are fabricated using microfluidics, loaded with 15 base-long secondary DNA targets, capped with a polyelectrolyte bilayer, and finally coated with a monolayer of polystyrene microspheres functionalized with duplexes comprised of immobilized probes and soluble, 13 base-long hybridization partner strands. Once warmed to 37 °C, secondary DNA targets are released from the gelatin template and then competitively displace the shorter, original hybridization partners on the polystyrene microspheres.

Exploring locked nucleic acids as a bio-inspired materials assembly and disassembly tool

Oligonucleotides hold great promise as a recognition-based biomaterials assembly and disassembly tool. Chemically modified oligonucleotides such as locked nucleic acids (LNA) provide the added advantage of nuclease resistance. In the current study, we focus on programming the assembly and disassembly of LNA-linked colloidal particles as a function of sequence composition. We find that incorporation of LNA residues (∼30%) into either one or both primary hybridization partner strands results in a higher duplex density than for isosequential DNA strands. Mismatched primary hybridization partners with sequence length of 11–15 bases have similar initial primary duplex densities. The displacement of mismatched strands by 15 base-long, perfectly matched competitive target strands, however, does depend on the base length of the original mismatched partner strand. Confocal microscopy confirms that substantial colloidal assembly occurs for both perfectly matched and mismatched LNA sequences that are 9 bases in length. Extensive disassembly for the mismatched case is then triggered through the introduction of 15 base-long competitive target strands. Our work demonstrates that LNA can be used to programmatically assemble and disassemble colloidal particles.

Azide photochemistry for facile modification of graphitic surfaces: preparation of DNA-coated carbon nanotubes for biosensing

A facile, two-step method for chemically attaching single-stranded DNA to graphitic surfaces, represented here by carbon nanotubes, is reported. In the first step, an azide-containing compound, N-5-azido-nitrobenzoyloxy succinimide (ANB-NOS), is used to form photo-adducts on the graphitic surfaces in a solid-state photochemical reaction, resulting in active ester groups being oriented for the subsequent reactions. In the second step, pre-synthesized DNA strands bearing a terminal amine group are coupled in an aqueous solution with the active esters on the photo-adducts. The versatility of the method is demonstrated by attaching pre-synthesized DNA to surfaces of carbon nanotubes in two platforms—as vertically-aligned multi-walled carbon nanotubes on a solid support and as tangled single-walled carbon nanotubes in mats. The reaction products at various stages were characterized by x-ray photoelectron spectroscopy. Two different assays were used to check that the DNA strands attached to the carbon nanotubes were able to bind their partner strands with complementary base sequences. The first assay, using partner DNA strands tethered to gold nanoparticles, enabled the sites of DNA attachment to the carbon nanotubes to be identified in TEM images. The second assay, using radioactively labelled partner DNA strands, quantified the density of functional DNA strands attached to the carbon nanotubes. The diversity of potential applications for these DNA-modified carbon-nanotube platforms is exemplified here by the successful use of a DNA-modified single-walled carbon-nanotube mat as an electrode for the specific detection of metal ions.

Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation.

DNMT1, the major maintenance DNA methyltransferase in animals, helps to regulate gene expression, genome imprinting, and X-chromosome inactivation. We report on the crystal structure of a productive covalent mouse DNMT1(731-1602)-DNA complex containing a central hemimethylated CpG site. The methyl group of methylcytosine is positioned within a shallow hydrophobic concave surface, whereas the cytosine on the target strand is looped out and covalently anchored within the catalytic pocket. The DNA is distorted at the hemimethylated CpG step, with side chains from catalytic and recognition loops inserting through both grooves to fill an intercalation-type cavity associated with a dual base flip-out on partner strands. Structural and biochemical data establish how a combination of active and autoinhibitory mechanisms ensures the high fidelity of DNMT1-mediated maintenance DNA methylation.

Down-regulation of miR-125a-3p in human gastric cancer and its clinicopathological significance

Recent reports have demonstrated that another strand of mature microRNA (miRNA), called microRNA* or 3p (5p) strand, which is generated from the same precursor miRNA (Pre-miR), has a crucial role in cellular function. We previously reported the tumor suppressive effect of miR-125a-5p in gastric cancer. The current study was designed to examine the function and clinical significance of miR-125a-3p, a partner strand of miR-125a-5p, in human gastric cancer. Quantitative RT-PCR was used to evaluate miR-125a-3p expression in 70 gastric cancer cases to determine the clinicopathologic significance of miR-125a-3p expression. In addition, the effect of miR-125a-3p on the proliferation of gastric cancer cells was investigated. Low expression levels of miR-125a-3p were associated with indicators of enhanced malignant potential such as tumor size (p=0.0002), tumor invasion (p=0.0149), lymph node metastasis (p=0.018), liver metastasis (p=0.016), peritoneal dissemination (p=0.03), advanced clinical stage (p=0.0037) and poor prognosis (p=0.0083). Multivariate analysis indicated that low miR-125a-3p expression was an independent prognostic factor for survival, while in vitro assays demonstrated that miR‑125a-3p suppressed the proliferation of gastric cancer cells. MiR-125a-3p is a potent prognostic marker in gastric cancer. The clinical significance and tumor suppressive effect of miR‑125a-3p, as well as previously reported miR-125a-5p, suggest that the functional role of another strand of the mature form miRNA cannot be ignored, at least in miR-125a biogenesis.