Complementary Base Sequences Research Articles

Abstract 3796: Exosomes enhance the radiation sensitivity via miR-6823-5p and modulate metastases in pancreatic cancer model

Abstract Introduction: Pancreatic cancer (PC) is believed to be a difficult disease because of its radio-resistance and metastases. Radiotherapy reported to modify tumor microenvironment (TME) and to affect PC progression. Exosomes containing such as microRNAs (miRNAs), messenger RNAs (mRNAs), and proteins of cells, play essential roles in cell-to-cell communications. However, characteristics and mechanisms for radiation responsive exosomes remain unclear. Here, we first report mechanisms of exosomes-mediated radiation response and regulation in PC metastasis with focusing on intra/intercellular communications via miRNA. Methods: MIAPaCa-2 cells, human pancreatic carcinoma, was used. To isolate exosomes, cell culture media was ultracentrifuged at 100,000 x g for 90 min at 4°C. Exosomes derived from non-irradiated culture media was named ‘0 Gy-Exo’ and those from irradiated with 5 Gy culture media is called ‘5 Gy-Exo’. Exosome sizes and features were evaluated by transmission electron microscopy (TEM). Intracellular reactive oxygen species (ROS) levels induced by exosomes and by X-ray irradiation were evaluated by the C-H2DCF dye staining. To detect DNA damage, cells were fixed and stained by γ-H2AX antibody. The expression of superoxide dismutase 1 (SOD1) was evaluated by using immunoblots. Total RNA was extracted from 0 Gy-Exo and 5 Gy-Exo, and then comprehensive miRNA expression analysis was performed to analyze 2,565 human miRNA sequences. The Gene Expression Omnibus database (GSE163133) and the TargetScan were used for specific miRNAs identification. Living MIAPaCa-2 cells in the presence or absence of those exosomes were injected in the spleen of Balb/c nude mice to establish liver metastatic models. Eight weeks after the injection, mice were sacrificed, and then the spleen and the liver were harvested as a whole organ. H-E staining and immunohisitochemical analyses using S100A4 and SMAD4 antibodies were performed to detect liver metastases. Results: Isolated exosomes were shaped in the form of closed, round vesicles with a diameter of 10~100 nm. Intracellular ROS levels and the numbers of γ-H2AX foci in the cells with 5 Gy-Exo were significantly increased compared to those the control (without adding exosomes, p<0.01). Intracellular SOD1 expression in the cells with the 5 Gy-Exo was decreased compared with that in the control. The miR-6823-5p was identified to have a complementary base sequence to SOD1. The average occupied proportions with MIAPaCa-2 cells in the liver was modulated according to those exosomes. The SMAD4 expression was highly observed in the liver and the spleen suggesting that the SMAD4 could be a useful marker of liver metastases. Conclusion: Exosomes increased radiation sensitivity through increase of intracellular ROS levels via exosomal miRNAs and also could affect liver metastases in pancreatic cancer models. Citation Format: Ai Nakaoka, Makiko Nakahana, Sachiko Inubushi, Yasuyuki Shimizu, Kazuma Iwashita, Naritoshi Mukumoto, Kana Kobayashi, Takeaki Ishihara, Daisuke Miyawaki, Ryohei Sasaki. Exosomes enhance the radiation sensitivity via miR-6823-5p and modulate metastases in pancreatic cancer model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3796.

Applications of Gene Therapy in Dentistry: A Review Article

AbstractGene therapy promises to possess a good prospect in bridging the gap between dental applications and medicine. The dynamic therapeutic modalities of gene therapy have been advancing rapidly. Conventional approaches are being revamped to be more comprehensive and pre-emptive, which could do away with the need for surgery and medicine altogether. The complementary base sequences known as genes convey the instructions required to manufacture proteins. The oral cavity is one of the most accessible locations for the therapeutic intervention of gene therapy for several oral tissues. In 1990, the first significant trial of gene therapy was overseen to alleviate adenosine deaminase deficiency. The notion of genetic engineering has become increasingly appealing as a reflection of its benefits over conventional treatment modalities. An example of how this technology may alter dentistry is the implementation of gene therapy for dental and oral ailments. The objective of this article is to examine the effects of gene therapy on the field of dentistry, periodontology and implantology. Furthermore, the therapeutic factors of disease therapy, minimal invasion, and appropriate outcome have indeed been taken into consideration.

Solvo-Driven Dimeric Nanoplasmon Coupling Under DNA Direction

Strong coupling is a prerequisite for nanoassemblies to deliver functions and applications unrealizable by noninteracting building units. DNA-Directed metamaterials, albeit highly programmable, oft...

Move blocked model predictive control with guaranteed stability and improved optimality using linear interpolation of base sequences

To mitigate the online computational load of model predictive control, move blocking, which parameterises either the input sequence or offset from the base sequence by fixing the decision variables over arbitrary time intervals, is commonly used. However, existing move blocking schemes use a fixed base sequence only and do not fully exploit the valuable properties from various base sequences. Thus, we propose the interpolated solution-based move blocking strategy which parameterises the offset from the convex combination of two complementary base sequences – infinite-horizon linear quadratic regulator solution and shifted previous solution – and optimises the interpolation parameter as an additional decision variable in the optimal control problem. This allows the controller to exploit the valuable properties from both solutions by choosing the optimal interpolation parameter and blocked offset according to the current state online. The proposed approach efficiently improves the optimality performance whileguaranteeing the recursive feasibility and closed-loop stability.

Online Capillary Electrophoresis Reaction for Interaction Study of Amino Acid Modified Peptide Nucleic Acid and Proteins

Abstract Peptide nucleic acid (PNA) is a nucleic acid analog which consists of purines, pyrimidines bases, and a neutrally charged peptide backbone. The PNA has the potential as a very useful biological probe for protein analysis since it has more in vivo biological stability as compared to DNA- or RNA-based aptamers. Usually, the addition of amino acids or peptide to the PNA backbone is used to improve its water-solubility and cell-permeability, but these modifications may affect the interaction between PNA and proteins. To date, the investigation of the interaction between PNA and proteins is rare, and there is no reported study about the effects of modifications. In this work, we designed two types of amino acid modified PNAs, (Lys)2-PNA and (Glu)2-PNA, which kept the same base sequence with 15-mer thrombin aptamer and had two basic lysine and two acidic glutamic acid residues on N-terminal of the peptide backbone, respectively. To rapidly assess the binding affinity and specificity of modified PNA and proteins, the online CE reaction method was developed to analyze the interactions of (Lys)2-PNA/(Glu)2-PNA and three proteins: thrombin (THB), single-strand DNA-binding protein (SSB) and human serum albumin (HSA). Meanwhile, the interactions of (Lys)2-PNA/(Glu)2-PNA and thrombin were compared with that of the corresponding complementary base sequence (Lys)2-cPNA/(Glu)2-cPNA and thrombin. The online CE reaction results showed that the interaction of (Lys)2-PNA and (Glu)2-PNA with three proteins was in the order of THB > SSB > HSA. However, (Lys)2-PNA and (Lys)2-cPNA showed similar binding affinity with thrombin; while the binding affinity of (Glu)2-PNA with thrombin was stronger than that of (Glu)2-cPNA with thrombin. Moreover, the binding constant Kb of (Glu)2-PNA and three proteins was determined by affinity capillary electrophoresis (ACE). The online CE reaction eliminates the requirement of incubation, and thus it is fast in detection, and easy to operate with minimum cost. The method is particularly suitable for the interaction studies of expensive modified PNAs and proteins, and can assist the design of PNA probe that binds to proteins.

Fabrication of Honeycomb Films Based on Aptamer and Binding Behavior Studies with Fluorophore-Labeled Complementary Base Sequences

A novel honeycomb film was successfully fabricated by self-assembly of polystyrene (PS)/polyethylene glycol (PEG)/nucleic acid aptamer with the assistance of dioctyl sodium sulfosuccinate (AOT) using the breath figure method. The nucleic acid aptamer embedded in the composite film remained bioactive and the film presented excellent fluorescent behaviors after binding with fluorophore-labeled complementary base sequences. The distribution of aptamer strands in the composite film was inferred from the fluorescence images and fluorescence spectra. The 3′ end and 5′ end of aptamer strands changed from being embedded to stretching out in the single pore of the composite film. The corresponding fluorescence images shifted from fluorescent spots to semicircular and annular fluorescent structures. This strategy can improve applications of honeycomb films in biosensors and microreactors.

MiRNAs as potential molecular targets in heart failure.

Pathogenesis of heart diseases is associated with an altered expression profile of hundreds of genes. miRNAs are a newly identified layer of gene regulation operating at the post-transcriptional level by pairing to complementary base sequences in target mRNAs. Genetic data have identified the roles of miRNAs in basic pathological processes associated with heart failure: apoptosis, fibrosis, myocardial hypertrophy and cardiac remodeling. Many reports demonstrated that aberrantly expressed miRNAs and their modulation have effects on cardiac insufficiency. Here, we overview the advances in miRNAs as potential targets in the modulation of the heart failure phenotype. miRNA-based therapy holds great promise as a future strategy for treating heart diseases and identifying emerging signaling pathways responsible for the progression of heart failure.

Peptide nucleic acid probe for protein affinity purification based on biotin–streptavidin interaction and peptide nucleic acid strand hybridization

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin–streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the “warhead”) and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl–PNA:PNA–biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons.

Research progress and applications of self-assembled DNA nanostructures

DNA nanotechnology is an example of bottom-up molecular self-assembly, in which molecular components spontaneously organize into stable structures; the particular form of these structures is induced by the physical and chemical properties of the components selected by the designers. In this field, nucleic acids are used as non-biological engineering materials for nanotechnology rather than as the carriers of genetic information in living cells. This use is enabled by the strict base pairing rules of nucleic acids, which cause only portions of strands with complementary base sequences to bind together to form strong, rigid double helix structures. This allows for the rational design of base sequences that will selectively assemble to form complex target structures with precisely controlled nanoscale features. This article mainly reviews the developments of DNA self-assembly achieved recently and also introduces the design methods of it, which would be helpful and enlightened for our future research.

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.

Preparation of supramolecular chromophoric assemblies using a DNA duplex

Organization of supramolecular assemblies of chromophores with precisely-controlled orientation and sequence remains challenging. Nucleic acids with complementary base sequences spontaneously form double-helical structures. Therefore, covalent attachment of chromophores to DNA or RNA can be used to control assembly and orientation of chromophores. In this perspective, we first review our recent work on the assemblies of fluorophores (pyrene and perylene) by using natural base pairs. The interaction between dyes can be strictly controlled by means of cluster and interstrand wedge motifs. We then discuss novel artificial base pairs that can suppress the interaction between fluorophores and nucleobases. We incorporated a cyclohexane moiety into DNA, and showed that these artificial base pairs suppressed the electron-hole transfer between fluorophores and nucleobases and enhanced the quantum yields of fluorophores. These base pairs can potentially be used to accumulate fluorophores inside DNA duplexes without decreasing quantum yields.

Artificial DNA Lattice Fabrication by Noncomplementarity and Geometrical Incompatibility

Fabrication of DNA nanostructures primarily follows two fundamental rules. First, DNA oligonucleotides mutually combine by Watson-Crick base-pairing rules between complementary base sequences. Second, the geometrical compatibility of the DNA oligonucleotide must match for lattices to form. Here we present a fabrication scheme of DNA nanostructures with noncomplementary and/or geometrically incompatible DNA oligonucleotides, which contradicts conventional DNA structure creation rules. Quantitative analyses of DNA lattice sizes were carried out to verify the unfavorable binding occurrences, which correspond to errors in algorithmic self-assembly. Further studies of these types of bindings may shed more light on the exact mechanisms at work in the self-assembly of DNA nanostructures.

Stabilization of DNA Multiassembly by Addition of a Phosphate Group at the 5′-Sticky End

Abstract The effect of 5′-phosphorylation at nicks on DNA stabilization was measured through the formation of DNA multiassemblies consisting of 40-mer half-sliding complementary oligonucleotides, which had overlapping complementary 20-mer base sequences. 5′-Phosphorylated oligonucleotides formed longer DNA multiassemblies than 5′-hydroxylated ones. This result indicated that the 5′-phosphorylated sticky ends contribute to the stabilization of the DNA association due to the hydrogen bonds between 3′-hydroxy groups and 5′-phosphate groups at the nick positions.

Small Volume Detection of Plasmodium Falciparum Using a 50 Micrometer Diameter Cavity with Self-contained Electrochemistry

An electrochemical enzyme-linked solid phase DNA-hybridization assay for the detection of Plasmodium falciparum is reported. The DNA-hybridization assay involves attachment of biotinylated primary DNA probe (10P) via its 5'-amine-terminus to the streptavidin- coated microwells in a 96-well plate. The 10P was used to capture the target by hybridization to a complementary sequence. A 36-mer secondary DNA probe labeled with alkaline phosphatase (20P~AP) on the 3'end was hybridized to a complementary base sequence on the 5' end of the target. The electrochemical detection involved the wall electrode (TNB, 8 μm2) as the detecting electrode and top layer gold (0.7 mm2 when 200 nL is placed on top of microcavity) as pseudoreference/auxiliary electrodes. The peak current from cyclic voltammograms of PAPP incubated inside modified microwells gave a linear relationship with the concentration of the target at 2- 50 ng/mL, where 10P and 20P~AP were 50 ng/mL. A detection limit of 1.4 ng/mL (or 70 pg) of the DNA target was obtained. The electrochemical signals were not affected in the presence of human hepatocytes, pig liver, or chicken serum indicating the viability of this assay in real clinical samples.

Hantavirus N Protein Exhibits Genus-Specific Recognition of the Viral RNA Panhandle

A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into "panhandle" hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5' and 3' ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.

Small-Volume Detection of Plasmodium falciparum CSP Gene Using a 50-μm-Diameter Cavity with Self-Contained Electrochemistry

An electrochemical enzyme-linked immobilized DNA-hybridization assay for the detection of Plasmodium falciparum has been developed. The target molecule was a segment of the repeat sequence of the gene coding for the circumsporozoite (CSP) protein from the AF54087 gene. This analyte offers the possibility of specifically detecting P. falciparum. The assay involves attachment of a biotinylated primary DNA probe via its 5'-amine-terminus to the streptavidin-coated surface of microwells in a 96-well plate. The primary DNA probe (1(0)P, which was of two different sequences we call 1(0)P(a) and 1(0)P(b)) was used to capture the target (T, which was of two different sequences, T1 sequence 481-590 and T2 sequence 472-590 of AF54087 gene for the CSP gene) by hybridization to a complementary sequence on the target. On 1(0)P(a), 47 bases were complementary to T1 and T2 at 543-590, while on 1(0)P(b), 35 bases were complementary to T1 and T2 at 555-590. A secondary DNA probe that contained 36 bases with alkaline phosphatase (2(0)P-AP) label on the 3' end was hybridized to a complementary base sequence on the 5' end of the target. p-Aminophenol, which is enzymatically generated by the immobilized AP from p-aminophenyl phosphate (PAPP), is detected using electrochemistry. The peak current of cyclic voltammograms from a PAPP solution incubated inside the microwells modified with the complete assembly of the assay components gives a linear relationship with the concentration of the target (2-50 ng/mL, where P1 (P1a and P1b) and P2-AP concentrations are 50 ng/mL). A detection limit of 1.4 ng/mL (or 46 pM) of the DNA target was obtained. The signals of the assays were not significantly affected when performed in the presence of human hepatocytes, pig liver, or chicken serum indicating the viability of this assay in real clinical samples.

Strategies for the efficient synthesis of stimuli-responsive ss-DNA bioconjugates for capture and transfection of plasmid DNA

The paper describes the preparation of a stimuli-responsive single stranded DNA-bioconjugate and its use for the capture and transient transfection of plasmid DNA. The two segments of the bioconjugate consisted of a thermo-responsive poly-( N-isopropylacrylamide) oligomer (mass average <4000 g/mol, polydispersity <1.3, critical solution temperature in pure water approximately 33 °C) and a homo-pyrimidine oligonucleotide sequence (ss 5′–(CTT) n –3′). The oligomer was produced by chain transfer polymerization in the presence of a suitable chain transfer agent and carried an O-alkylhydroxylamine, respectively, a hydrazide end group. Direct chemical ligation was possible with the oxidized form of the DNA oligonucleotide bearing an additional riboU at the 3′-end. With this single step reaction similar DNA coupling rates (up to 80%) could be achieved as by an established protocol based on the use of a vinyl sulphone-activated oligomer with a thiol-modified oligonucleotide. The bioconjugate was able to selectively extract double stranded plasmid DNA bearing a complementary base sequence from bacterial lysate (recovery yields between 70% and 80%, purity >99%) by triple helix affinity interaction/precipitation. The complex could be directly transfected into mammalian cells (transient transfection, Lipofectamine protocol) with success rates similar or superior to those obtained with pure plasmid DNA obtained by conventional plasmid preparation kits.

Inducing Reversible Stiffness Changes in DNA-crosslinked Gels

Researchers have constructed a number of DNA-based nanodevices that undergo stepped configuration changes through the application of single-stranded DNA oligomers. Such devices can be incorporated into gel networks to create new classes of active materials with controllable bulk mechanical properties. This concept was demonstrated in a DNA-crosslinked gel, the stiffness of which was modulated through the application of DNA strands. Each crosslink incorporated a single-stranded region to which a DNA strand with a complementary base sequence (called the fuel strand) bound, thereby changing the nanostructure of the gel network. The gel was restored to its initial stiffness through the application of the complement of the fuel strand, which cleared the fuel strand from the crosslink via competitive binding. Stiffness changes in excess of a factor of three were observed. The ability to switch the mechanical properties of these gels without changing temperature, buffer composition, or other environmental conditions, apart from the application of DNA, makes these materials attractive candidates for biotechnology applications.

Visualization of DNA by Scanning Probe Microscopy.

Using the pulse injection method, single-stranded DNA and double-stranded plasmid DNA have been deposited on well-defined Cu (111) surfaces under ultra-high vacuum (UHV) condition to obtain high resolution scanning tunneling microscopy (STM) images. These particular UHV-STM images have revealed that DNA molecules are adsorbed directly onto a clean Cu (111) surface, and exhibited the detailed structures of DNA which have not been resolved earlier. The single-stranded DNA oligomers have exhibited the images of individual internal base molecules and helix structures made of complementary base sequence. For the double-stranded plasmid DNA, the images has visualized Watson-Crick double-helix structures.

High-resolution scanning tunneling microscopy imaging of DNA molecules on Cu(111) surfaces

Using the pulse injection method, single-stranded DNA and double-stranded plasmid DNA have been deposited on well-defined Cu(111) surfaces under ultrahigh vacuum (UHV) conditions to obtain high-resolution scanning tunneling microscope (STM) images. These particular UHV-STM images have revealed that DNA molecules are adsorbed directly onto a clean Cu(111) surface and exhibited the detailed structures of DNA, which has not been resolved previously. The single-stranded DNA oligomers have exhibited the images of individual internal base molecules and the helix structures made of complementary base sequences. For the double-stranded plasmid DNA, the images have shown the Watson–Crick double-helix structure.