Incorporation Of Nucleobases Research Articles

A Nucleobase‐Driven Self‐Gelled Hyaluronic Acid‐Based Injectable Adhesive Hydrogel Enhances Intervertebral Disc Repair

AbstractCell therapy strategies hold great promise for nucleus pulposus (NP) regeneration and intervertebral disc degeneration (IVDD) treatment. However, their therapeutic efficiency is compromised by the anoikis of administered cells and the harsh environment in degenerated intervertebral discs (IVD). Inspired by the ability of nucleobases to form multiple H‐bonds, a nucleobase‐driven self‐gelling strategy is proposed for the incorporation of nucleobases (e.g., thymine) into hyaluronic acid (HA) chains to trigger gel formation through enhanced intermolecular H‐bond interactions. An aqueous solution of a thymine‐modified HA (HAT) supramolecular polymer is shown to exhibit self‐gelation behavior, injectability, tissue adhesion, and hydration capacity (comparable to that observed in the NP tissue of a 25‐year‐old adult). These characteristics enable the injection and filling of the HAT hydrogel system in the IVD, integration of the separated NP tissues, restoration of the biomechanical functions of the degenerated IVD, and securing of the encapsulated cells in the NP site. Manganese dioxide (MnO2) nanoparticles incorporated into the HAT system (HATMn) regulate oxidative stress and the hypoxic microenvironment in degenerated IVD, ensuring the viability of the encapsulated cells. The 12‐week in vivo study results demonstrate that the administration of the MSCs‐laden HATMn system (HATMn‐MSCs) effectively restores the structure and function of degenerated IVD.

Mechanical characterization of bio composite films as a novel drug carrier platform for sustained release of 5-fluorouracil for colon cancer: Methodological investigation

In this study, we employed Pectin (PC) as a matrix that is hybridized with three different nucleobase (NB) units (cytosine, thymine, uracil) to generate pectin–nucleobase(PC-NB) biocomposite films stabilized through bio-multiple hydrogen bonds (BMHBs) as drug carrier for anticancer 5-Fluorouracil (5-FU). Prepared biocomposite films were characterized by Fourier Transform Infra-red Spectroscopy (FTIR), X-ray Diffraction (XRD), Thermogravimmetry Analysis (TGA) and Scanning Electron Microscope (SEM). Mechanical and sorption properties were also evaluated. In vitro drug release performed in both acidic pH 1.2 (stomach pH) and alkaline pH 7.4 (intestinal pH) showed that incorporation of nucleobases into pectin significantly restricted release rate of 5-FU particularly under acidic condition (pH 1.2). Hemolysis assays demonstrated that PC-NB-5-FU biocomposite film drug carriers were hemocompatible. Confocal microscope analysis indicates facilitated cellular uptake of PC-NB-5-FU film in HT-29 colon cancer cell line, which in turn result in a higher potential of apoptosis. Confocal imaging of fluorescent live/dead cell indicators and MTT assay outcomes, both demonstrated significant decreases in cellular viability of PC-NB-5-FU biocomposite films. Collectively, our findings indicate that this PC-NB-5-FU biocomposite films can be conferred as a proficient formulation for targeted delivery of colon cancer drugs.

Scalable Synthesis, In Vitro cccDNA Reduction, and In Vivo Antihepatitis B Virus Activity of a Phosphonomethoxydeoxythreosyl Adenine Prodrug.

Standard literature procedures for the chemical synthesis of l-threose nucleosides generally employ l-ascorbic acid as starting material. Herein, we have explored two alternative routes that start from either l-arabitol or l-diethyl tartrate, both affording 2-O-methyl-l-threofuranose as a key building block for nucleobase incorporation. The access to multigram quantities of this glycosyl donor in a reproducible fashion allows for the preparation of 2'-deoxy-α-l-threofuranosyl phosphonate nucleosides on a large scale. This methodology was applied to the gram scale synthesis of an aryloxy amidate prodrug of phosphonomethoxydeoxythreosyl adenine. This prodrug exerted potent activity against an entecavir-resistant hepatitis B virus (HBV) strain, while leading to a significant reduction in the levels of HBV covalently closed circular DNA in a cellular assay. Furthermore, its remarkable anti-HBV efficacy was also confirmed in vivo using a hydrodynamic injection-based HBV mouse model, without relevant toxicity and systemic exposure occurring.

Building better enzymes: Molecular basis of improved non-natural nucleobase incorporation by an evolved DNA polymerase.

Obtaining semisynthetic microorganisms that exploit the information density of “hachimoji” DNA requires access to engineered DNA polymerases. A KlenTaq variant has been reported that incorporates the “hachimoji” P:Z nucleobase pair with a similar efficiency to that seen for Watson–Crick nucleobase incorporation by the wild type (WT) KlenTaq DNA polymerase. The variant polymerase differs from WT KlenTaq by only four amino acid substitutions, none of which are located within the active site. We now report molecular dynamics (MD) simulations on a series of binary complexes aimed at elucidating the contributions of the four amino acid substitutions to altered catalytic activity. These simulations suggest that WT KlenTaq is insufficiently flexible to be able to bind AEGIS DNA correctly, leading to the loss of key protein/DNA interactions needed to position the binary complex for efficient incorporation of the “hachimoji” Z nucleobase. In addition, we test literature hypotheses about the functional roles of each amino acid substitution and provide a molecular description of how individual residue changes contribute to the improved activity of the KlenTaq variant. We demonstrate that MD simulations have a clear role to play in systematically screening DNA polymerase variants capable of incorporating different types of nonnatural nucleobases thereby limiting the number that need to be characterized by experiment. It is now possible to build DNA molecules containing nonnatural nucleobase pairs in addition to A:T and G:C. Exploiting this development in synthetic biology requires engineered DNA polymerases that can replicate nonnatural nucleobase pairs. Computational studies on a DNA polymerase variant reveal how amino acid substitutions outside of the active site yield an enzyme that replicates nonnatural nucleobase pairs with high efficiency. This work will facilitate efforts to obtain bacteria possessing an expanded genetic alphabet.

Synthesis and Conformation of Pentopyranoside Nucleoside Phosphonates.

In contrast to natural nucleosides, where the nucleobase is positioned at the anomeric center, we report the synthesis of pentopyranoside nucleosides with a phosphonate functionality at the 1'-anomeric oxygen. Starting from l-arabinose, key functionalized l- glycero- and l- erythro-pentopyranose carbohydrate synthons were prepared and further elaborated into the final six-membered ring nucleosides via nucleobase incorporation and phosphonomethylation reactions. NMR analysis demonstrated that these nucleoside phosphonates exist in solution as conformers predominantly adopting a chair structure in which the base moiety is equatorially positioned. Such conformation prevents unfavorable 1,3-diaxial steric and electronic interactions. Notably, the stereochemical outcome of the Vorbrüggen glycosylation step utilized en route to the thymine analogue clearly suggests the absence of anchimeric assistance, as opposed to what is usually observed during nucleoside synthesis using protected furanose precursors. The finding that the diphosphates of the compounds developed in this study are recognized by DNA polymerases is important in view of the future selection of artificial genetic systems and dedicated polymerases as well as applications in therapy.

Acetyl-protected cytosine and guanine containing acrylics as supramolecular adhesives

ABSTRACTHydrogen bonding among nucleobase pairs serves as an efficient noncovalent interaction for designing supramolecular polymers with desired properties for pressure sensitive adhesives. Michael addition yielded acetyl-protected cytosine/guanine containing acrylic monomers with flexible spacers between the hydrogen bonding units and the acrylic backbone. Free radical polymerization of nucleobase-containing monomers afforded acetyl-protected cytosine/guanine homopolymers and random copolymers with n-butyl acrylate. Nucleobase incorporation significantly affected thermal, thermomechanical, rheological, morphological properties, and adhesive performance of polyacrylates. Guanine/cytosine-containing copolymers each exhibited a single glass transition (Tg) that increased with increasing nucleobase content. Self-association of acetyl cytosine and acetyl guanine units converted low Tg polyacrylates to physically crosslinked networks with mechanical integrity. Solution casting acetyl guanine-containing copolymers with 8 mol% or higher guanine content yielded free-standing films with microphase-separated morphologies. Acetyl cytosine-containing copolymers with 15 mol% or more cytosine formed free-standing films with less microphase-separation compared to the guanine copolymers. 1H NMR titration experiments established a 1:1 binding stoichiometry between acetyl cytosine and acetyl guanine monomers in CDCl3, similar to guanine-cytosine association. However, the acetyl protecting group hindered the formation of triple hydrogen bonding, resulting in double hydrogen bonding between acetyl cytosine and acetyl guanine with an intermediate binding strength comparable to their self-associations. Acetyl guanine-containing copolymers with 3 mol% acetyl guanine exhibited higher peel strength on stainless steel and higher extended service frequency range compared to cytosine-containing copolymers and various pressure sensitive adhesive controls.

Site-Specific Covalent Labeling of RNA by Enzymatic Transglycosylation.

We demonstrate the site-specific incorporation of nucleobase derivatives bearing fluorophores or affinity labels into a short RNA stem loop recognition motif by exchange of a guanine residue. The RNA-TAG (transglycosylation at guanosine) is carried out by a bacterial (E. coli) tRNA guanine transglycosylase (TGT), whose natural substrate is the nitrogenous base PreQ1. Remarkably, we have successfully incorporated large functional groups including biotin, BODIPY, thiazole orange, and Cy7 through a polyethylene glycol linker attached to the exocyclic amine of PreQ1. Larger RNAs, such as mRNA transcripts, can be site-specifically labeled if they possess the 17-nucleotide hairpin recognition motif. The RNA-TAG methodology could facilitate the detection and manipulation of RNA molecules by enabling the direct incorporation of functional artificial nucleobases using a simple hairpin recognition element.

Core-crosslinked amphiphilic biodegradable copolymer based on the complementary multiple hydrogen bonds of nucleobases: synthesis, self-assembly and in vitro drug delivery

Nucleobases (adenine and thymine) were conjugated to the amphiphilic biodegradable copolymers methoxyl poly(ethylene glycol)-b-poly(L-lactide-co-2-methyl-2-allyloxycarbonylpropylene carbonate) (mPEG-b-P(LA-co-MAC)). The hydrogen bonds between adenine (A) and thymine (T) were confirmed by 1H NMR titration experiments and FT-IR. It was found that the incorporation of nucleobases into the hydrophobic segment of the amphiphilic copolymers could be used for core-crosslinking of the formed micelles containing a lowered critical micelle concentration (CMC) via hydrogen bond interaction between A and T in aqueous solution. The anticancer drug doxorubicin (DOX) was encapsulated into the copolymer micelles. The in vitro drug release profile showed that the incorporation of nucleobases significantly restricted DOX release at pH 7.4, because of the compact crosslinking structure of micelles. However, a much faster release rate was observed at pH 5.0, due to the dissociation of hydrogen bonds between nucleobases. This character facilitates drug delivery in the acidic tumor micro-environment inside the endosome. Meanwhile, the DOX-loaded core-cross-linked micelles could be efficiently internalized into cancer cells and exhibit similar anticancer efficacy as free DOX against MDA-MB-231 cells. Therefore, the complementary multiple hydrogen bonds of nucleobases provided a convenient tool to stabilize the micelle structures by forming core-crosslinking, and could be further applied for controlled drug delivery.

Access to Oxetane-Containing psico-Nucleosides from 2-Methyleneoxetanes: A Role for Neighboring Group Participation?

The first psico-oxetanocin analogue of the powerful antiviral natural product, oxetanocin A, has been readily synthesized from cis-2-butene-1,4-diol. Key 2-methyleneoxetane precursors were derived from β-lactones prepared by the carbonylation of epoxides. F(+)-mediated nucleobase incorporation provided the corresponding nucleosides in good yield but with low diastereoselectivity. Surprisingly, attempted exploitation of anchimeric assistance to increase the selectivity was not fruitful. A range of 2-methyleneoxetane and related 2-methylenetetrahydrofuran substrates was prepared to explore the basis for this. With one exception, these substrates also showed little stereoselectivity in nucleobase incorporation. Computational studies were undertaken to examine if neighboring group participation involving fused [4.2.0] or [4.3.0] intermediates is favorable.

A Rapid Synthetic Route to Conformationally Restricted [5,5]-Bicyclic Nucleoside-Amino Acid Conjugates

A concise synthetic route to a novel class of conformationally rigid 3 ′,4 ′-cis-fused bicyclic nucleoside derivatives has been developed. The synthetic strategy and approach involves initial synthesis of a key [5,5]-bicyclic 6-aminofurofuran-2-one scaffold, employing an L-serine derived aminobutenolide as a strategically functionalized chiral template. Subsequent utilization of the carbonyl functionality of the above bicyclic lactone toward nucleobase incorporation, and linking of the resident amine functionality with appropriately protected amino acids completed the syntheses of the target bicyclic nucleoside-amino acid conjugates. Following the above route, and utilizing a combination of easily available nucleobases (4) and amino acids (4) as the two diversity elements, combinatorial synthesis of a 16-member demonstration library of the title amino acid-linked nucleosides has been accomplished.

Stepwise Translocation of Dpo4 Polymerase during Error-Free Bypass of an oxoG Lesion

7,8-dihydro-8-oxoguanine (oxoG), the predominant lesion formed following oxidative damage of DNA by reactive oxygen species, is processed differently by replicative and bypass polymerases. Our kinetic primer extension studies demonstrate that the bypass polymerase Dpo4 preferentially inserts C opposite oxoG, and also preferentially extends from the oxoG•C base pair, thus achieving error-free bypass of this lesion. We have determined the crystal structures of preinsertion binary, insertion ternary, and postinsertion binary complexes of oxoG-modified template-primer DNA and Dpo4. These structures provide insights into the translocation mechanics of the bypass polymerase during a complete cycle of nucleotide incorporation. Specifically, during noncovalent dCTP insertion opposite oxoG (or G), the little-finger domain–DNA phosphate contacts translocate by one nucleotide step, while the thumb domain–DNA phosphate contacts remain fixed. By contrast, during the nucleotidyl transfer reaction that covalently incorporates C opposite oxoG, the thumb-domain–phosphate contacts are translocated by one nucleotide step, while the little-finger contacts with phosphate groups remain fixed. These stepwise conformational transitions accompanying nucleoside triphosphate binding and covalent nucleobase incorporation during a full replication cycle of Dpo4-catalyzed bypass of the oxoG lesion are distinct from the translocation events in replicative polymerases.

Incorporation of deoxyribonucleosides into DNA of coryneform bacteria and the relevance of deoxyribonucleoside kinases.

In order to obtain basic knowledge of the salvage pathways for DNA synthesis, the ability of Brevibacterium ammoniagenes ATCC 6872 and Micrococcus luteus ATCC 15932 for incorporation of nucleobases and nucleosides was investigated. Only adenine and uracil are incorporated by B. ammoniagenes, whereas M. luteus additionally can utilize deoxyadenosine and, less efficiently, thymidine. In M. luteus, the demonstration of deoxyadenosine kinase and thymidine kinase explains the incorporation data. Uptake of thymidine is of short duration because of rapid breakdown of exogenously supplied thymidine to thymine. At a 540-fold excess pyrimidine deoxyribonucleosides inhibit 14C incorporation from thymidine nearly totally and purine deoxyribonucleosides cut by half the uptake rate, probably by interfering with transport of thymidine. However, as no cessation of thymidine incorporation occurs at these concentrations of purine deoxyribonucleosides, incorporation is finally enhanced. During the initial period of this reduced uptake considerable protection of thymidine from breakdown to thymine is provided by deoxyguanosine, but not by deoxyadenosine. At a 108-fold excess there is actually no inhibition of thymidine uptake by deoxyguanosine and only an insignificant impairment by deoxyadenosine resulting in an ultimate enhancement of 14C incorporation up to 20% of the exogenously supplied thymidine. As there is no salvage pathway for thymidine in B. ammoniagenes due to the absence of thymidine kinase, labelling with adenine and hydrolyzing of the 'contaminated' RNA fraction with 1 M KOH is recommended for measurements of overall DNA synthesis in this strain.