Oligodeoxynucleotides containing formamidopyrimidine lesions and C-nucleoside analogues at defined sites were prepared by solid-phase synthesis and in some cases enzymatic ligation. Formamidopyrimidine lesions were introduced as dinucleotides to prevent rearrangement to their pyranose isomers. Oligodeoxynucleotides containing single diastereomers of C-nucleoside analogues of Fapy.dA were introduced by using the respective phosphoramidites. The formamidopyrimidine lesions reduce the T(M) of dodecamers relative to their unmodified nucleotide counterparts when opposite the nucleotide proper base-pairing partner. However, duplexes containing Fapy.dG-dA mispairs melt significantly higher than those comprised of dG-dA. All duplexes containing Fapy.dA-dX or its C-nucleoside analogue melt lower than the respective complexes containing dA-dX. Studies of the alkaline lability of oligodeoxynucleotides containing formamidopyrimidine lesions indicate that Fapy.dA is readily identified as an alkali-labile lesion with use of piperidine (1.0 M, 90 degrees C, 20 min), but Fapy.dG is less easily identified in this manner.

We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5′‐protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA‐target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off‐to‐on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.

Biomolecule immobilization has attracted the attention of various fields such as fine chemistry and biomedicine for their use in several applications such as wastewater, immunosensors, biofuels, et cetera. The performance of immobilized biomolecules depends on the substrate and the immobilization method utilized. Electrospun nanofibers act as an excellent substrate for immobilization due to their large surface area to volume ratio and interconnectivity. While biomolecules can be immobilized using adsorption and encapsulation, covalent immobilization offers a way to permanently fix the material to the fiber surface resulting in high efficiency, good specificity, and excellent stability. This review aims to highlight the various covalent immobilization techniques being utilized and their benefits and drawbacks. These methods typically fall into two categories: (1) direct immobilization and (2) use of crosslinkers. Direct immobilization techniques are usually simple and utilize the strong electrophilic functional groups on the nanofiber. While crosslinkers are used as an intermediary between the nanofiber substrate and the biomolecule, with some crosslinkers being present in the final product and others simply facilitating the reactions. We aim to provide an explanation of each immobilization technique, biomolecules commonly paired with said technique and the benefit of immobilization over the free biomolecule.

Fapy.dG is produced in DNA as a result of oxidative stress. Under some conditions Fapy.dG is formed in greater yields than 8-oxodG from a common chemical precursor. Recently, Fapy.dG and its C-nucleoside analogue were incorporated in chemically synthesized oligonucleotides at defined sites. Like 8-oxodG, Fapy.dG instructs DNA polymerase to misincorporate dA opposite it in vitro. The interactions of DNA containing Fapy.dG or the nonhydrolyzable analogue with Fpg and MutY are described. Fpg excises Fapy.dG (K(M) = 2.0 nM, k(cat) = 0.14 min(-1)) opposite dC approximately 17-fold more efficiently than when mispaired with dA, which is misinserted by DNA polymerase in vitro. Fpg also prefers to bind duplexes containing Fapy.dG.dC or beta-C-Fapy.dG.dC compared to those in which the lesion is opposite dA. MutY incises dA when it is opposite Fapy.dG and strongly binds duplexes containing the lesion or beta-C-Fapy.dG. Incision from Fapy.dG.dA is faster than from dG.dA mispairs but slower than from DNA containing 8-oxodG opposite dA. These data demonstrate that Fapy.dG closely resembles the interactions of 8-oxodG with two members of the GO repair pathway in vitro. The similar effects of Fapy.dG and 8-oxodG on DNA polymerase and repair enzymes in vitro raise the question as to whether Fapy.dG elicits similar effects in vivo.

Human mitochondrial methionine transfer RNA (hmtRNAMetCAU) has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position-34 (f5C34). The role of this modification in (hmtRNAMetCAU) for the decoding of AUA, as well as AUG, in both the peptidyl- and aminoacyl-sites of the ribosome in either chain initiation or chain elongation is still unknown. We report the first synthesis and analyses of the tRNA's anticodon stem and loop domain containing the 5-formylcytidine modification. The modification contributes to the tRNA's anticodon domain structure, thermodynamic properties and its ability to bind codons AUA and AUG in translational initiation and elongation.

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNAMetCAU, to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl-(A-) sites of the ribosome. The hmtRNAMetCAU has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f5C34), and a cytidine substituting for the invariant uridine at position 33 of the canonical “U-turn” in tRNAs. The structure of the tRNA's anticodon stem and loop domain (hmtASLMetCAU), determined by NMR restrained molecular modeling, revealed how the f5C34 modification facilitates the decoding of AUA at the P- and A-sites. The f5C34 defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASLMetCAU. The hmtASLMetCAU exhibited a “C-turn” conformation that has some characteristics of the U-turn motif. Codon binding studies with both E. coli and bovine mitochondrial ribosomes revealed that the f5C34 facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL's binding kinetics. Mitochondrial translation by many organisms including humans sometimes initiates with the universal isoleucine codons AUU and AUC. The f5C34 enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f5C34, expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria.

The formamidopyrimidines Fapy.dA and Fapy.dG are produced in DNA as a result of oxidative stress. These lesions readily epimerize in water, an unusual property for nucleosides. The equilibrium mixture slightly favors the beta-anomer, but the configurational status in DNA is unknown. The ability of endonuclease IV (Endo IV) to efficiently incise alpha-deoxyadenosine was used as a tool to determine the configuration of Fapy.dA and Fapy.dG in DNA. Endo IV incision of the C-nucleoside analogues of Fapy.dA was used to establish selectivity for the alpha-anomer. Incision of alpha-C-Fapy.dA follows Michaelis-Menten kinetics (K(m) = 144.0 +/- 7.5 nM, k(cat) = 0.58 +/- 0.21 min(-1)), but the beta-isomer is a poor substrate. Fapy.dA incision is considerably slower than that of alpha-C-Fapy.dA, and does not proceed to completion. Endo IV incision of Fapy.dA proceeds further upon rehybridization, suggesting that the lesion reequilibrates and that the enzyme preferentially cleaves duplex DNA containing alpha-Fapy.dA. The extent of Fapy.dA incision suggests that the lesion exists predominantly ( approximately 90%) as the beta-anomer in DNA. Endo IV incises Fapy.dG to less than 5% under comparable reaction conditions, suggesting that the lesion exists almost exclusively as its beta-anomer in DNA.

We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5′‐protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA‐target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off‐to‐on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.