scholarly journals Mechanistic analysis of the enhanced RNAi activity by 6-mCEPh-purine at the 5’ end of the siRNA guide strand

RNA ◽  
2020 ◽  
pp. rna.073775.119
Author(s):  
Vincent Brechin ◽  
Fumikazu Shinohara ◽  
Jun-ichi Saito ◽  
Herve Seitz ◽  
Yukihide Tomari
Keyword(s):  
Guide Strand ◽  
Mechanistic Analysis ◽  
Rnai Activity

scholarly journals siRNAs containing 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2′-F-NMC NTPs

2021 ◽  
Author(s):  
Masaaki Akabane-Nakata ◽  
Namrata D Erande ◽  
Pawan Kumar ◽  
Rohan Degaonkar ◽  
Jason A Gilbert ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Mitochondrial Rna ◽  
Seed Region ◽  
Guide Strand ◽  
Passenger Strand ◽  
Molecular Modeling Studies ◽  
Rnai Activity ◽  
Rna And Dna

Abstract We recently reported the synthesis of 2′-fluorinated Northern-methanocarbacyclic (2′-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2′-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2′-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5′ phosphate, suggesting that the 2′-F-NMC is a poor substrate for 5′ kinases. In mice, the 2′-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2′-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5′-phosphate mimic 5′-(E)-vinylphosphonate was attached to the 2′-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2′-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2′-F-NMC. Finally, the 5′-triphosphate of 2′-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.

scholarly journals Investigation of Strand-Selective Interaction of SNA-Modified siRNA with AGO2-MID

2020 ◽  
Vol 21 (15) ◽  
pp. 5218 ◽  
Author(s):  
Yukiko Kamiya ◽  
Yuuki Takeyama ◽  
Tomonari Mizuno ◽  
Fuminori Satoh ◽  
Hiroyuki Asanuma
Keyword(s):  
Nucleic Acid ◽  
Inhibitory Effect ◽  
Guide Strand ◽  
Argonaute 2 ◽  
Passenger Strand ◽  
Nuclease Resistance ◽  
Interfering Rna ◽  
Target Activity ◽  
Rnai Activity ◽  
Target Effects

Small interfering RNA (siRNA) has been recognized as a powerful gene-silencing tool. For therapeutic application, chemical modification is often required to improve the properties of siRNA, including its nuclease resistance, activity, off-target effects, and tissue distribution. Careful siRNA guide strand selection in the RNA-induced silencing complex (RISC) is important to increase the RNA interference (RNAi) activity as well as to reduce off-target effects. The passenger strand-mediated off-target activity was previously reduced and on-target activity was enhanced by substitution with acyclic artificial nucleic acid, namely serinol nucleic acid (SNA). In the present study, the reduction of off-target activity caused by the passenger strand was investigated by modifying siRNAs with SNA. The interactions of SNA-substituted mononucleotides, dinucleotides, and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-labeled double-stranded RNA (dsRNA) with the MID domain of the Argonaute 2 (AGO2) protein, which plays a pivotal role in strand selection by accommodation of the 5’-terminus of siRNA, were comprehensively analyzed. The obtained nuclear magnetic resonance (NMR) data revealed that AGO2-MID selectively bound to the guide strand of siRNA due to the inhibitory effect of the SNA backbone located at the 5’ end of the passenger strand.

Naphthoquinones and derivatives for chemotherapy: perspectives and limitations of their anti-trypanosomatids activities

2020 ◽  
Vol 26 ◽  
Author(s):  
Luíza Dantas-Pereira ◽  
Edézio F. Cunha-Junior ◽  
Valter V. Andrade-Neto ◽  
John F. Bower ◽  
Guilherme A. M. Jardim ◽  
...  
Keyword(s):  
Large Scale ◽  
Neglected Tropical Diseases ◽  
Folk Medicine ◽  
Leishmania Species ◽  
Parasitic Infections ◽  
Mechanistic Analysis ◽  
Leishmania Spp ◽  
Nanomolar Range

: Chagas disease, Sleeping sickness and Leishmaniasis, caused by trypanosomatids Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively, are considered neglected tropical diseases, and they especially affect impoverished populations in the developing world. The available chemotherapies are very limited and a search for alternatives is still necessary. In folk medicine, natural naphthoquinones have been employed for the treatment of a great variety of illnesses, including parasitic infections. This review is focused on the anti-trypanosomatid activity and mechanistic analysis of naphthoquinones and derivatives. Among all the series of derivatives tested in vitro, naphthoquinone-derived 1,2,3-triazoles were very active on T. cruzi infective forms in blood bank conditions, as well as in amastigotes of Leishmania spp. naphthoquinones containing a CF3 on a phenyl amine ring inhibited T. brucei proliferation in the nanomolar range, and naphthopterocarpanquinones stood out for their activity on a range of Leishmania species. Some of these compounds showed a promising selectivity index (SI) (30 to 1900), supporting further analysis in animal models. Indeed, high toxicity to the host and inactivation by blood components are crucial obstacles to be overcome to use naphthoquinones and/or their derivatives for chemotherapy. Multidisciplinary initiatives embracing medicinal chemistry, bioinformatics, biochemistry, and molecular and cellular biology need to be encouraged to allow the optimization of these compounds. Large scale automated tests are pivotal for the efficiency of the screening step, and subsequent evaluation of both the mechanism of action in vitro and pharmacokinetics in vivo are essential for the development of a novel, specific and safe derivative, minimizing adverse effects.

scholarly journals PPM1G promotes the progression of hepatocellular carcinoma via phosphorylation regulation of alternative splicing protein SRSF3

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Dawei Chen ◽  
Zhenguo Zhao ◽  
Lu Chen ◽  
Qinghua Li ◽  
Jixue Zou ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Alternative Splicing ◽  
Protein Function ◽  
Vital Role ◽  
Normal Tissues ◽  
Mechanistic Analysis ◽  
Highly Correlated ◽  
Splicing Patterns

AbstractEmerging evidence has demonstrated that alternative splicing has a vital role in regulating protein function, but how alternative splicing factors can be regulated remains unclear. We showed that the PPM1G, a protein phosphatase, regulated the phosphorylation of SRSF3 in hepatocellular carcinoma (HCC) and contributed to the proliferation, invasion, and metastasis of HCC. PPM1G was highly expressed in HCC tissues compared to adjacent normal tissues, and higher levels of PPM1G were observed in adverse staged HCCs. The higher levels of PPM1G were highly correlated with poor prognosis, which was further validated in the TCGA cohort. The knockdown of PPM1G inhibited the cell growth and invasion of HCC cell lines. Further studies showed that the knockdown of PPM1G inhibited tumor growth in vivo. The mechanistic analysis showed that the PPM1G interacted with proteins related to alternative splicing, including SRSF3. Overexpression of PPM1G promoted the dephosphorylation of SRSF3 and changed the alternative splicing patterns of genes related to the cell cycle, the transcriptional regulation in HCC cells. In addition, we also demonstrated that the promoter of PPM1G was activated by multiple transcription factors and co-activators, including MYC/MAX and EP300, MED1, and ELF1. Our study highlighted the essential role of PPM1G in HCC and shed new light on unveiling the regulation of alternative splicing in malignant transformation.

Sign in / Sign up

Export Citation Format

Share Document