Dual Crosslinking Photo-Switches for Orthogonal Photo-Control of Hybridization Between Serinol Nucleic Acid and RNA.

Wavelength-selective photo-regulation by multiple chromophores responding to different wavelengths can expand the variation of photo-manipulating systems. Herein, we report the orthogonal photo-regulation of duplex formation between serinol nucleic acid (SNA) and RNA using light-induced crosslinking reactions mediated by a new photo-reactive nucleobase 8-naphthylvinyladenine (NV A) and previously described 8-pyrenylvinyladenine (PV A). An intrastrand crosslink was induced in an SNA strand containing two adjacent NV A residues by irradiation with 340-405 nm light; the crosslink was reversed by irradiation with ≤300 nm light. In an SNA strand with adjacent NV A and PV A residues, an intrastrand crosslink resulted from irradiation with 405-465 nm light that was reversed by irradiation with ≤340 nm light. Intrastrand photo-crosslinking caused severe destabilization of an SNA/RNA duplex, resulting in dissociation to single strands. Cycloreversion resulted in duplex formation. With these NV A/NV A and NV A/PV A photo-switches, four hybridization states of two SNA/RNA duplexes could be orthogonally photo-controlled by irradiation with a suitable wavelength of light.

DNA damage and repair following traumatic brain injury.

Traumatic brain injury (TBI) is known to promote significant DNA damage irrespective of age, sex, and species. Chemical as well as structural DNA modification start within minutes and persist for days after TBI. Although several DNA repair pathways are induced following TBI, the simultaneous downregulation of some of the genes and proteins of these pathways leads to an aberrant overall DNA repair process. In many instances, DNA damages escape even the most robust repair mechanisms, especially when the repair process becomes overwhelmed or becomes inefficient by severe or repeated injuries. The persisting DNA damage and/or lack of DNA repair contributes to long-term functional deficits. In this review, we discuss the mechanisms of TBI-induced DNA damage and repair. We further discussed the putative experimental therapies that target the members of the DNA repair process for improved outcome following TBI.

High Enrichment of Nucleobase-modified Aptamers in Early Selection Rounds by Microbeads-assisted Capillary Electrophoresis SELEX.

Nucleobase-modified aptamers are attractive candidates for diagnostic and therapeutic agents due to the high affinity, stability and functionality. However, since even conventional SELEX requires many selection rounds, acquisition of modified aptamers is much more laborious. Herein, microbeads-assisted capillary electrophoresis (MACE)-SELEX was applied against thrombin using the indole-modified DNA library. After only three selection rounds, we successfully enriched the modified aptamers and they showed slower off-rate than reported aptamers, suggesting MACE-SELEX is a promising approach for rapid identification of modified aptamers.

Copper-Catalyzed Alkyne-Azide Cycloaddition on the Solid Phase for the Preparation of Fully Click-Modified Nucleic Acids.

Click chemistry has become a widely used method to insert modifications into DNA. Due to its commercial availability, 5'-ethynyl-deoxyuridine (EdU) is commonly incorporated into the DNA for subsequent modification by click reaction. However, it is partially oxidized during deprotection during solid-phase synthesis, resulting in a ketone that is no longer accessible for click modification. To enable the high-fidelity solid-phase synthesis of EdU-containing DNA, this protocol describes a procedure to perform the click reaction on the solid phase before deprotection. Afterwards, the DNA can be deprotected and purified according to standard procedures, and the full modification of EdU with the azide of choice can be analyzed by HPLC and HPLC/MS.

Azobenzene-bridged diradical janus nucleobases with photo-converted magnetic properties between antiferromagnetic and ferromagnetic couplings.

We computationally design a series of azobenzene (AB)-bridged double radicalized nucleobases, a novel kind of diradical Janus-type nucleobases, and explore their spin coupling characteristics. Calculations prove that such diradical Janus-bases not only normally match with their complementary bases, but also exhibit well-defined diradical character with photo-convertible intramolecular magnetic couplings (antiferromagnetic vs. ferromagnetic). Combination of four radical nucleobases (rG, rA, rC, rT) and photoswitch AB can yield 10 diradical Janus-bases with different magnetic characteristics in which AB functions a bridge to mediate the spin coupling between two radical bases. The trans-form supports mild antiferromagnetic couplings with the spin coupling constants (J) ranging from -153.6 cm-1 to -50.91 cm-1 while the cis-form has weak magnetic couplings with ferromagnetic (0.22-8.50 cm-1 ) for most of them or antiferromagnetic (-0.77, -1.73, -3.30 cm-1 ) properties for only three. Further structural examination and frontier molecular orbital analyses indicate that the extended π conjugation for better spin polarization provides an effective through-π-bond pathway to mediate the spin coupling in the trans conformation while nonplanarity of the cis conformation weakens the through-bond coupling and causes a competitive through-space pathway and as an overall result inhibits the spin coupling between two spin moieties. Meanwhile, we also find that the J values of the cis conformation vary with their angle between the radical base and its linked phenylene. Furthermore, the magnetic properties of the diradical Janus-bases can be significantly increased by interacting with metal ions. They also maintain a good UV absorption characteristics and there is a clear redshift compared with AB. This work provides a promising strategy for the rational design of photo-convertible Janus-base magnets as the magnetism-tunable DNA building blocks. © 2018 Wiley Periodicals, Inc.

New synthetic route to ethynyl-dUTP: A means to avoid formation of acetyl and chloro vinyl base-modified triphosphates that could poison SELEX experiments.

5-Ethynyl-2'-deoxyuridine is a common base-modified nucleoside analogue that has served in various applications including selection experiments for potent aptamers and in biosensing. The synthesis of the corresponding triphosphates involves a mild acidic deprotection step. Herein, we show that this deprotection leads to the formation of other nucleoside analogs which are easily converted to triphosphates. The modified nucleoside triphosphates are excellent substrates for numerous DNA polymerases under both primer extension and PCR conditions and could thus poison selection experiments by blocking sites that need to be further modified. The formation of these nucleoside analogs can be circumvented by application of a new synthetic route that is described herein.

Polymerase incorporation of a 2'-deoxynucleoside-5'-triphosphate bearing a 4-hydroxy-2-mercaptobenzimidazole nucleobase analogue.

Here, we describe the enzymatic construction of a new larger base pair formed between adenine (A) and a 4-hydroxy-2-mercaptobenzimidazole (SB) nucleobase analogue. We investigated the enzymatic incorporation of 2'-deoxynucleoside-5'-triphosphate bearing a SB nucleobase analogue (dSBTP) into oligonucleotides (ONs) by DNA polymerases. dSBTP could be effectively incorporated at the site opposite a dA in a DNA template by several B family DNA polymerases. These findings provide new insights into various aspects of biotechnology, including the design of non-natural base pairs.

Base modification strategies to modulate immune stimulation by an siRNA.

Immune stimulation triggered by siRNAs is one of the major challenges in the development of safe RNAi-based therapeutics. Within an immunostimulatory siRNA sequence, this hurdle is commonly addressed by using ribose modifications (e.g., 2'-OMe or 2'-F), which results in decreased cytokine production. However, as immune stimulation by siRNAs is a sequence-dependent phenomenon, recognition of the nucleobases by the trigger receptor(s) is also likely. Here, we use the recently published crystal structures of Toll-like receptor 8 (TLR8) bound to small-molecule agonists to generate computational models for ribonucleotide binding by this immune receptor. Our modeling suggested that modification of either the Watson-Crick or Hoogsteen face of adenosine would disrupt nucleotide/TLR8 interactions. We employed chemical synthesis to alter either the Watson-Crick or Hoogsteen face of adenosine and evaluated the effect of these modifications in an siRNA guide strand by measuring the immunostimulatory and RNA interference properties. For the siRNA guide strand tested, we found that modifying the Watson-Crick face is generally more effective at blocking TNFα production in human peripheral blood mononuclear cells (PBMCs) than modification at the Hoogsteen edge. We also observed that modifications near the 5'-end were more effective at blocking cytokine production than those placed at the 3'-end. This work advances our understanding of how chemical modifications can be used to optimize siRNA performance.