scholarly journals Site-Selective Artificial Ribonucleases: Oligonucleotide Conjugates Containing Multiple Imidazole Residues in the Catalytic Domain

2011 ◽  
Vol 2011 ◽  
pp. 1-17 ◽  
Author(s):  
Natalia G. Beloglazova ◽  
Martin M. Fabani ◽  
Nikolai N. Polushin ◽  
Vladimir V. Sil'nikov ◽  
Valentin V. Vlassov ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Artificial Ribonucleases ◽  
Phosphodiester Bond ◽  
Cleavage Activity ◽  
Anchor Group ◽  
Rna Targeting ◽  
Challenging Tasks ◽  
Nucleotide Residue ◽  
Site Selective ◽  
Oligonucleotide Conjugates

Design of site-selective artificial ribonucleases (aRNases) is one of the most challenging tasks in RNA targeting. Here, we designed and studied oligonucleotide-based aRNases containingmultipleimidazole residues in the catalytic part and systematically varied structure of cleaving constructs. We demonstrated that the ribonuclease activity of the conjugates is strongly affected by the number of imidazole residues in the catalytic part, the length of a linker between the catalytic imidazole groups of the construct and the oligonucleotide, and the type of anchor group, connecting linker structure and the oligonucleotide. Molecular modeling of the most active aRNases showed that preferable orientation(s) of cleaving constructs strongly depend on the structure of the anchor group and length of the linker. The inclusion of deoxyribothymidine anchor group significantly reduced the probability of cleaving groups to locate near the cleavage site, presumably due to a stacking interaction with the neighbouring nucleotide residue. Altogether the obtained results show that dynamics factors play an important role in site-specific RNA cleavage. Remarkably high cleavage activity was displayed by the conjugates with the most flexible and extended cleaving construct, which presumably provides a better opportunity for imidazole residues to be correctly positioned in the vicinity of scissile phosphodiester bond.

Manipulation of Single-Stranded DNA by Using an Artificial Site-Selective DNA Cutter Composed of Cerium(IV)/EDTA and Phosphonate-Oligonucleotide Conjugates

2011 ◽  
Vol 6 (9) ◽  
pp. 2407-2411 ◽  
Author(s):  
Yuichiro Aiba ◽  
Tuomas Lönnberg ◽  
Makoto Komiyama
Keyword(s):  
Single Stranded Dna ◽  
Site Selective ◽  
Oligonucleotide Conjugates

scholarly journals Design and Synthesis of Peptide-Oligonucleotide Conjugates as Potential Artificial Ribonucleases

2008 ◽  
Vol 52 (1) ◽  
pp. 529-530
Author(s):  
I. Serpokrylova ◽  
L. Koroleva ◽  
N. Svischeva ◽  
D. Novopashina ◽  
V. Silnikov
Keyword(s):  
Artificial Ribonucleases ◽  
Design And Synthesis ◽  
Oligonucleotide Conjugates

Factor XII Osaka: Abnormal factor XII with partially defective prekallikrein cleavage activity

2011 ◽  
Vol 105 (03) ◽  
pp. 473-478 ◽  
Author(s):  
Yuya Arakawa ◽  
Yuya Sugahara ◽  
Michiko Matsushita ◽  
Yuki Moriguchi ◽  
Hisashi Shimohiro ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Factor Xii ◽  
Charged Surface ◽  
Antigen Level ◽  
Chromogenic Substrates ◽  
Cleavage Activity ◽  
Normal Ranges ◽  
Japanese Male ◽  
Normal Individuals ◽  
Exon 10

SummaryA healthy Japanese male had reduced factor XII (FXII) activity (35%) in contrast to normal antigen levels (81%). The F12 of this proband had a 9775G to C mutation in exon 10 and an 11276G to A mutation in exon 13 that resulted in two amino acid substitutions of Ala324Pro (GCG→CCG) in the proline-rich connecting region and Gly531Glu (GGG→GAG) near the active Ser544 in the catalytic domain. His father had the nucleotide 46T/T and a heterozygous 9775G/C mutation. The FXII activity (32%) and antigen level (38%) of the father were about half that of normal individuals with 46T/T, suggesting a heterozygous cross reacting material (CRM)-negative deficiency. His mother had a 46C/T and heterozygous 11276G/A mutation, and 80% FXII activity was incompatible with the corresponding antigen level (125%), suggesting a heterozygous CRM-positive deficiency. The substitution of Ala324Pro probably caused the CRM-negative mutation and the Gly531Glu caused the CRM-positive mutation. We developed three methods based on chromogenic substrates to assay the distinct functions of FXII, namely its autoactivation on a negatively charged surface, activation by kallikrein cleavage and the prekallikrein cleavage activity of FXIIa. The ratios of autoactivated FXIIa/FXII antigen (0.80–1.10) and of kallikrein-induced FXIIa/FXII antigen (0.86–1.00) in plasma from the proband were within normal ranges, whereas those of FXIIa-induced kallikrein/FXII antigen were reduced to 0.41–0.45. In conclusion, the 9775G to C and 11276G to A mutations of F12 led to a CRM-negative and -positive FXII deficiency, and the F12 with 11276A produced a dys-functional type of FXII with a partial defect (0.41–0.45) in prekallikrein cleavage activity.

Site-Selective Artificial Ribonucleases and their Applications

2007 ◽  
Vol 11 (16) ◽  
pp. 1450-1459 ◽  
Author(s):  
Akinori Kuzuya ◽  
Makoto Komiyama
Keyword(s):  
Artificial Ribonucleases ◽  
Site Selective

scholarly journals Site-Selective Artificial Ribonucleases: Renaissance of Oligonucleotide Conjugates for Irreversible Cleavage of RNA Sequences

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1732
Author(s):  
Yaroslav Staroseletz ◽  
Svetlana Gaponova ◽  
Olga Patutina ◽  
Elena Bichenkova ◽  
Bahareh Amirloo ◽  
...  
Keyword(s):  
Metal Ion ◽  
Protein Complexes ◽  
Artificial Ribonucleases ◽  
Rna Sequences ◽  
Molecular Scaffold ◽  
Catalytically Active ◽  
Recognition Motifs ◽  
The One ◽  
Site Selective

RNA-targeting therapeutics require highly efficient sequence-specific devices capable of RNA irreversible degradation in vivo. The most developed methods of sequence-specific RNA cleavage, such as siRNA or antisense oligonucleotides (ASO), are currently based on recruitment of either intracellular multi-protein complexes or enzymes, leaving alternative approaches (e.g., ribozymes and DNAzymes) far behind. Recently, site-selective artificial ribonucleases combining the oligonucleotide recognition motifs (or their structural analogues) and catalytically active groups in a single molecular scaffold have been proven to be a great competitor to siRNA and ASO. Using the most efficient catalytic groups, utilising both metal ion-dependent (Cu(II)-2,9-dimethylphenanthroline) and metal ion-free (Tris(2-aminobenzimidazole)) on the one hand and PNA as an RNA recognising oligonucleotide on the other, allowed site-selective artificial RNases to be created with half-lives of 0.5–1 h. Artificial RNases based on the catalytic peptide [(ArgLeu)2Gly]2 were able to take progress a step further by demonstrating an ability to cleave miRNA-21 in tumour cells and provide a significant reduction of tumour growth in mice.

scholarly journals CasRx-mediated RNA targeting prevents choroidal neovascularization in a mouse model of age-related macular degeneration

2020 ◽  
Author(s):  
Changyang Zhou ◽  
Xinde Hu ◽  
Cheng Tang ◽  
Wenjia Liu ◽  
Shaoran Wang ◽  
...  
Keyword(s):  
Mouse Model ◽  
Macular Degeneration ◽  
Choroidal Neovascularization ◽  
Age Related Macular Degeneration ◽  
Adeno Associated Virus ◽  
Cleavage Activity ◽  
Age Related ◽  
Family Protein ◽  
Rna Targeting

AbstractThe smallest Cas13 family protein, CasRx, has a high cleavage activity and targeting specificity, offering attractive opportunity for therapeutic applications. Here we report that delivery of CasRx by adeno-associated virus via intravitreal injection could efficiently knockdown Vegfa transcripts and significantly reduce the area of laser-induced choroidal neovascularization in a mouse model of age-related macular degeneration. Thus, RNA-targeting CRISPR system could be used for in vivo gene therapy.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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