Structural investigation of functional nucleic acids

DNA enzymes, also known as deoxyribozymes, are synthetic single-stranded DNA molecules able to catalyze chemical reactions. There are two main reasons for studying deoxyribozymes: their practical value in various applications, and the understanding of basic properties - such as folding and catalysis - of a biopolymer that is of central importance for life. Compared to ribozymes, the DNA enzymes have a potential value as tools for industrial or therapeutic applications, owing to more cost-effective synthesis and higher stability. The first crystal structure of a deoxyribozyme demonstrated that DNA possesses the intrinsic ability to adopt complex tertiary folds that support catalysis and unveiled the active site of a DNA enzyme in the post-catalytic state (Ponce-Salvatierra, Wawrzyniak-Turek et al. 2016). The second reported crystal structure of the RNA-cleaving deoxyribozyme complements observations about the folds and catalysis of DNA enzymes although the structure was derived with DNA as a substrate mimic of RNA (Liu, Yu et al. 2017). These crystal structures represent a breakthrough in the field, but they are still insufficient to derive a clear mechanistic picture of the specific features of different RNA ligating and RNA cleaving deoxyribozymes. Therefore, ongoing efforts are devoted to structurally investigating additional deoxyribozymes. The new DNA enzymes were evolved to discriminate modified and unmodified RNA substrates and provide attractive tools for studying the natural epitranscriptomic RNA modification N6-methyladenosine (Sednev, Mykhailiuk et al. 2018). In the present study, the goal is to elucidate the structural basis for recognition of the methylated nucleobase by solving the crystal structure of the m6A sensitive RNA-cleaving deoxyribozyme in complex with an uncleavable analog of the RNA substrate, containing either methylated and unmethylated adenosine. Surprisingly, the RNA substrate dissociated from the deoxyribozyme during the crystallization process. Two structures for unmethylated and one of the methylated RNA substrate analog were solved. The next goal is to elucidate the crystal structure of the RNA-ligating deoxyribozyme in the pre-catalytic state of reaction. The previously reported crystal structure of the 9DB1 in the post-catalytic state of reaction could not explain the role of magnesium cations as cofactors for accelerating RNA ligation and properly describe the ligation mechanism. The structural investigation of the 9DB1 in the pre-catalytic state resulted in the ligation of the two RNA substrates during the crystallization process. In the future, other strategies are necessary to solve the questions on substrate recognition and catalytic mechanism of the RNA-cleaving and RNA-ligating deoxyribozymes investigated in this study. The second project deals with synthetic RNA aptamers that were identified by in vitro selection to mimic fluorescent proteins for RNA imaging and the development of biosensors. Several examples of fluorogen-activating RNA aptamers are known, and for some, the crystal structures have recently been solved e.g. of the Spinach, Mango, and Corn aptamers, that bind synthetic analogs of the GFP chromophore (Neubacher and Hennig 2019). The Chili is a new fluorogenic-RNA aptamer that mimics large Stokes shift (LSS) fluorescent proteins (FPs) by inducing highly Stokes‐shifted emission from several new green and red HBI (4-hydroxybenzylidene imidazolinone) derivatives that are non‐fluorescent when free in solution (Steinmetzger, Palanisamy et al. 2019). The new fluorophores are the first variants of fluorogenic aptamer ligands with permanently cationic sidechains that are bound by the RNA in their protonated phenol form, while emission occurs from the phenolate intermediate after excited-state proton transfer. The Chili–DMHBO+ complex is the longest wavelength-emitting (592 nm) and tightest binding (KD=12 nM) RNA fluorophore currently known in the growing family of HBI-binding aptamers. By employing X-ray crystallography, I have elucidated the three-dimensional structure of the Chili fluorophore binding site and revealed the structural basis for the large apparent Stokes shift and the promiscuity of the Chili aptamer to activate red and green-emitting chromophores2022-03-2

Pharmacological versus genetic inhibition of heme oxygenase-1 : the comparison of metalloporphyrins, shRNA and CRISPR/Cas9 system

Inhibition of heme oxygenase-1 (HO-1, encoded by HMOX1), a cytoprotective, anti-apoptotic and anti-inflammatory enzyme, may serve as a valuable therapy in various pathophysiological processes, including tumorigenesis. We compared the effect of chemical inhibitors - metalloporphyrins, with genetic tools - shRNA and CRISPR/Cas9 systems, to knock-down (KD)/knock-out (KO) HO-1 expression/activity. 293T cells were incubated with metalloporphyrins, tin and zinc protoporphyrins (SnPPIX and ZnPPIX, respectively) or were either transduced with lentiviral vectors encoding different shRNA sequences against HO-1 or were modified by CRISPR/Cas9 system targeting HMOX1. Metalloporphyrins decreased HO activity but concomitantly strongly induced HO-1 mRNA and protein in 293T cells. On the other hand, only slight basal HO-1 inhibition in shRNA KD 293T cell lines was confirmed on mRNA and protein level with no significant effect on enzyme activity. Nevertheless, silencing effect was much stronger when CRISPR/Cas9-mediated knock-out was performed. Most of the clones harboring mutations within HMOX1 locus did not express HO-1 protein and failed to increase bilirubin concentration after hemin stimulation. Furthermore, CRISPR/Cas9-mediated HO-1 depletion decreased 293T viability, growth, clonogenic potential and increased sensitivity to H2O2 treatment. In summary, we have shown that not all technologies can be used for inhibition of HO activity in vitro with the same efficiency. In our hands, the most potent and comprehensible results can be obtained using genetic tools, especially CRISPR/Cas9 approach

Tuneable helices of plasmonic nanoparticles using liquid crystal templates: molecular dynamics investigation of an unusual odd–even effect in liquid crystalline dimers

Liquid crystalline (LC) dimers formed helical nanofilaments depending on the parity of the alkyl linker, revealing an unusual odd–even effect. Molecular dynamics simulations were used to investigate the observed tendency. Elongation of the linker translates to an increase of the pitch of the helices, which allows achieving tuneable helical assemblies of Au nanoparticles doped to the LC matrix. The impact of the tuneable pitch of helices on the chiral optical properties of composites was investigated with full-wave simulations based on the T-matrix method

Structure and mechanism of the methyltransferase ribozyme MTR1

RNA-catalysed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyses the site-specific synthesis of 1-methyladenosine (m$^1$A) in RNA, using O$^6$-methylguanine (m$^6$G) as methyl group donor. Here we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2'-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution

Size-Dependent Thermo- and Photoresponsive Plasmonic Properties of Liquid Crystalline Gold Nanoparticles

Achieving remotely controlled, reversibly reconfigurable assemblies of plasmonic nanoparticles is a prerequisite for the development of future photonic technologies. Here, we obtained a series of gold-nanoparticle-based materials which exhibit long-range order, and which are controlled with light or thermal stimuli. The influence of the metallic core size and organic shell composition on the switchability is considered, with emphasis on achieving light-responsive behavior at room temperature and high yield production of nanoparticles. The latter translates to a wide size distribution of metallic cores but does not prevent their assembly into various, switchable 3D and 2D long-range ordered structures. These results provide clear guidelines as to the impact of size, size distribution, and organic shell composition on self-assembly, thus enhancing the smart design process of multi-responsive nanomaterials in a condensed state, hardly attainable by other self-assembly methods which usually require solvents

(S)-2-(4-Chlorobenzoyl)-1,2,3,4-tetrahydrobenzo[e]pyrazino[1,2-a][1,4]diazepine-6,12(11H,12aH)-dione—Synthesis and Crystallographic Studies

(S)-2-(4-Chlorobenzoyl)-1,2,3,4-tetrahydrobenzo[e]pyrazino[1,2-a][1,4]diazepine-6,12(11H,12aH)-dione was obtained in a three-step, one-pot synthesis, starting from optically pure (S)-2-piperazine carboxylic acid dihydrochloride. Selective acylation of the β-nitrogen atom followed by condensation with isatoic anhydride and cyclization with HATU/DIPEA to a seven-member benzodiazepine ring, led to the tricyclic benzodiazepine derivative. Crystallographic studies and initial biological screening were performed for the title compound

Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine

Fluorogenic RNA aptamers such as Chili display strong fluorescence enhancement upon aptamer–ligand complex formation. Here, the authors provide insights into the mechanism of fluorescence activation of Chili by solving the crystal structures of Chili with its bound positively charged ligands DMHBO+ and DMHBI+, and they reveal that Chili uses an excited state proton transfer mechanism based on time-resolved optical spectroscopy measurements

Novel (S)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12(2H,11H)-dione derivatives: Selective inhibition of MV-4-11 biphenotypic B myelomonocytic leukemia cells’ growth is accompanied by reactive oxygen species overproduction and apoptosis

A series of optically pure (R)- and (S)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12(2H,11H)-dione derivatives was designed and synthesized as novel anthramycin analogues in a three-step, one-pot procedure, and tested for their antiproliferative activity on nine following cell lines: MV-4-11, UMUC-3, MDA-MB-231, MCF7, LoVo, HT-29, A-549, A2780 and BALB/3T3. The key structural features responsible for exhibition of cytotoxic effect were determined: the (S)-configuration of chiral center and the presence of hydrophobic 4-biphenyl substituent in the side chain. Introduction of bromine atom into the 8 position (8g) or substitution of dilactam ring with benzyl group (8m) further improved the activity and selectivity of investigated compounds. Among others, compound 8g exhibited selective cytotoxic effect against MV-4-11 (IC50 = 8.7 μM) and HT-29 (IC50 = 17.8 μM) cell lines, while 8m showed noticeable anticancer activity against MV-4-11 (IC50 = 10.8 μM) and LoVo (IC50 = 11.0 μM) cell lines. The cell cycle arrest in G1/S checkpoint and apoptosis associated with overproduction of reactive oxygen species was also observed for 8e and 8m

Allogenic Adipose-Derived Stem Cells in Diabetic Foot Ulcer Treatment: Clinical Effectiveness, Safety, Survival in the Wound Site, and Proteomic Impact

Although encouraging results of adipose-derived stem cell (ADSC) use in wound healing are available, the mechanism of action has been studied mainly in vitro and in animals. This work aimed to examine the safety and efficacy of allogenic ADSCs in human diabetic foot ulcer treatment, in combination with the analyses of the wound. Equal groups of 23 participants each received fibrin gel with ADSCs or fibrin gel alone. The clinical effects were assessed at four time points: days 7, 14, 21 and 49. Material collected during debridement from a subset of each group was analyzed for the presence of ADSC donor DNA and proteomic changes. The reduction in wound size was greater at all subsequent visits, significantly on day 21 and 49, and the time to 50% reduction in the wound size was significantly shorter in patients who received ADSCs. Complete healing was achieved at the end of the study in seven patients treated with ADSCs vs. one treated without ADSCs. One week after ADSC application, 34 proteins significantly differentiated the material from both groups, seven of which, i.e., GAPDH, CAT, ACTN1, KRT1, KRT9, SCL4A1, and TPI, positively correlated with the healing rate. We detected ADSC donor DNA up to 21 days after administration. We confirmed ADSC-related improvement in wound healing that correlated with the molecular background, which provides insights into the role of ADSCs in wound healing—a step toward the development of cell-based therapies