The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNALys3UUU as the primer for reverse transcription. NCp7 also remodels the htRNA’s amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNA’s unique composition of post-transcriptional modifications on NCp7 recognition of htRNALys3UUU, the protein’s binding and functional remodeling of the human anticodon stem and loop domain (hASLLys3) were studied. NCp7 bound the hASLLys3UUU modified with 5-methoxycarbonyl methyl-2-thiouridine at position-34 (mcm5s2U34) and 2-methylthio-N6-threonylcarbamoyladenosine at position-37 (ms2t6A37) with a considerably higher affinity than the unmodified hASLLys3UUU (Kd = 0.28 ± 0.03 and 2.30 ± 0.62 μM, respectively). NCp7 denatured the structure of the hASLLys3UUU-mcm5s2U34;ms2t6A37;ψ39 more effectively than that of the unmodified hASLLys3UUU. Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average Kd = 0.55 ± 0.10 μM) and specificity for the ASLLys3UUU-mcm5s2U34;ms2t6A37 comparable to that of NCp7. The peptides recognized a t6A37-modified ASL with an affinity (Kd = 0.60 ± 0.09 μM) comparable to that for hASLLys3UUU-mcm5s2U34;ms2t6A37, indicating a preference for the t6A37 modification. Significantly, one of the peptides was capable of relaxing the hASLLys3UUU-mcm5s2U34;ms2t6A37;ψ39 structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNALys3UUU have been found to be important determinants of NCp7’s recognition prior to the tRNALys3UUU being annealed to the viral genome as the primer of reverse transcription.

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Background Most studies on regenerative medicine focus on cell-based therapies and transplantations. Small-molecule therapeutics, though proved effective in different medical conditions, have not been extensively investigated in regenerative research. It is known that healing potential decreases with development and developmental changes are driven by epigenetic mechanisms, which suggests epigenetic repression of regenerative capacity. Methods We applied zebularine, a nucleoside inhibitor of DNA methyltransferases, to stimulate the regenerative response in a model of ear pinna injury in mice. Findings We observed the regeneration of complex tissue that was manifested as improved ear hole repair in mice that received intraperitoneal injections of zebularine. Six weeks after injury, the mean hole area decreased by 83.2 ± 9.4% in zebularine-treated and by 43.6 ± 15.4% in control mice (p < 10 −30 ). Combined delivery of zebularine and retinoic acid potentiated and accelerated this effect, resulting in complete ear hole closure within three weeks after injury. We found a decrease in DNA methylation and transcriptional activation of neurodevelopmental and pluripotency genes in the regenerating tissues. Interpretation This study is the first to demonstrate an effective induction of complex tissue regeneration in adult mammals using zebularine. We showed that the synergistic action of an epigenetic drug (zebularine) and a transcriptional activator (retinoic acid) could be effectively utilized to induce the regenerative response, thus delineating a novel pharmacological strategy for regeneration. The strategy was effective in the model of ear pinna regeneration in mice, but zebularine acts on different cell types, therefore, a similar approach can be tested in other tissues and organs.

Many biologically active compounds, including macromolecules that are used as various kinds of drugs, must be delivered to the interior of cell or organelles such as mitochondria or nuclei to achieve a therapeutic effect. However, very often, lipophilic cell membrane is impermeable for these molecules. A new method in the transport of macromolecules through the cell membrane is the one based on utilizing cell-penetrating peptides (CPPs). Invented 25 years ago, CPPs are currently the subject of intensive research in many laboratories all over the world. CPPs are short compounds comprising up to 30 amino acid residues, which penetrate the cell membrane but do not cause cell damage. Additionally, CPPs can transfer hydrophilic molecules (peptides, proteins, nucleic acids) which exceed their mass, and for which the cell membrane is generally impermeable. In this review, we concentrate on the cellular uptake mechanism of CPPs and a method of conjunction of CPPs to the transported molecules. We also highlight the potential of CPPs in delivering various kinds of macromolecules into cells, including compounds of therapeutic interest.