22-nt端粒RNA片段所形成反向平行G-四链体的构型影响要素分析

不同构象的G-四链体通常展现出与蛋白质结合能力不同,进而发挥出不同的生理功能,分析G-四链体的构型影响要素,对探究其生理功能有重要意义。不同于DNA,RNA序列在以往的研究中均呈现平行G-四链体结构,前期研究中我们发现并报道了目前唯一能形成反向平行G-四链体的RNA序列,22-nt端粒RNA片段(ORN-N,A_(1)G_(2)G_(3)G_(4)U_(5)U_(6)A_(7)G_(8)G_(9)G_(10)U_(11)U_(12)A_(13)G_(14)G_(15)G_(16)U_(17)U_(18)A_(19)G_(20)G_(21)G_(22))。分析ORN-N的构型影响因素将极大拓展对RNA特性的认识。因此,本研究旨在系统性的探索能够影响ORN-N构型的因素。使用圆二色谱和熔融曲线监测糖环2′-羟基对ORN-N构型和稳定性的影响,发现ORN-N中不同核苷糖环2′-羟基对于反向平行G-四链体的作用不同。U_(6)、A_(7)、G_(16)的糖环2′-羟基对ORN-N的拓扑构象-维持至关重要,G_(3)、A_(13)、A_(19)的糖环2′-羟基具有稳定其构象的作用,而U_(17)的糖环2′-羟基降低了ORN-N的稳定性,此与以往研究中糖环2′-羟基不利于RNA序列形成反向平行G-四链体构型不同。将ORN-N环中的核苷U进行5-甲基化修饰,通过圆二色谱和熔融曲线监测其构象和稳定性,发现环中部分U碱基5-甲基化能稳定ORN-N的构象。使用5%~40%PEG模拟细胞内分子拥挤环境,通过圆二色谱监测其构象,结果与以往研究不同,分子拥挤环境并不能稳定反向平行RNA G-四链体,相反ORN-N的构象发生了翻转。综上,在ORN-N中糖环2′-羟基、环序列甲基化以及分子拥挤环境对反向平行RNA G-四链体的构型具有极其重要的影响。

Evolutionary selection on synonymous codons in RNA G-quadruplex structural region

To investigate how synonymous codons have been adapted to the formation of ribonucleic acid(RNA)G-quadruplex(rG4)structure,a computational searching algorithm G4Hunter was applied to detect rG4 structures in protein-coding sequences of mRNAs in five eukaryotic species.The native sequences forming rG4s were then compared with randomized sequences to evaluate selection on synonymous codons.Factors that may influence the formation of rG4 were also investigated,and the selection pressures of rG4 in different gene regions were compared to explore its potential roles in gene regulation.The results show universal selective pressure acts on synonymous codons in rG4 regions to facilitate rG4 formation in five eukaryotic organisms.While G-rich codon combinations are preferred in the rG4 structural region,C-rich codon combinations are selectively unfavorable for rG4 formation.Gene's codon usage bias,nucleotide composition,and evolutionary rate can account for the selective variations on synonymous codons among rG4 structures within a species.Moreover,rG4 structures in the translational initiation region showed significantly higher selective pressures than those in the translational elongation region.

Evolution of Monkeypox C9L RNA G-Quadruplex Elevates the Translation of Its Only Kelch-like Protein by Accelerating G-Quadruplex Unfolding Kinetics

Genomic surveillance of monkeypox virus(MPXV)is essential to explore the reason of its unusual outbreak.Current phylogenomic analysis of the MPXV genome mainly focuses on the effect of amino acid mutations.Herein,we explore the evolutionary variation of RNA G-quadruplex(RG4)of MPXV and find that the genome evolution of MPXV can also produce new effects through changes in the RG4 structure.This RG4 is located in MPXV’s only Kelch-like C9L gene,which encodes for an antagonist of the innate immune response.The evolution of this virus increases the unfolding kinetic constant of C9L RG4 and promotes the C9 protein level in living cells.Importantly,all reported MPXV genomes in 2022 carry the C9L-RG4-5 pattern with the highest unfolding kinetic constant.Additionally,the RG4 ligand,RGB-1,can impede the unfolding of C9L-RG4-5 and thereby reduce the C9 protein level.These findings carve out a new path to comprehensively understanding MPXV virology.

Fluorescent Visualization of Nucleolar G-Quadruplex RNA and Dynamics of Cytoplasm and Intranuclear Viscosity

The nucleolus,the locus of ribosome biogenesis,was found to be the predominant intracellular target of a new fluorescent probe,V-P1.In solution,the probe demonstrated both a selectivity to RNA G-quadruplexes and a sensitivity to the viscosity,while G-quadruplex binding did not disturb the viscosity sensing.In cells,confocal and fluorescence lifetime imaging,combined with digestion and competition experiments,lent support to the hypothesis of an RNA-based G-quadruplex as the intracellular target,postulated to be nucleolar ribosomal RNA(rRNA).The probe demonstrated a high sensitivity to viscosity in both the cytoplasm and the nuclear compartment and was used to precisely interrogate the viscosity changes resulting from diverse stimuli,such as temperature,monensin treatment,and etoposide-induced apoptosis.Owing to the putative rRNA G-quadruplex binding in vitro and in vivo,and further combined with a relatively low degree of toxicity,the dye enabled the interrogation of cytoplasm and intranuclear viscosity changes under diverse conditions and found applications in studying the influence and significance of cytoplasm and intranuclear viscosity as well as in gaining insight into the native secondary structure of rRNA in nucleoli.

5′-tiRNA-Gln inhibits hepatocellular carcinoma progression by repressing translation through the interaction with eukaryotic initiation factor 4A-I

tRNA-derived small RNAs(tsRNAs)are novel non-coding RNAs that are involved in the occurrence and progression of diverse diseases.However,their exact presence and function in hepatocellular carcinoma(HCC)remain unclear.Here,differentially expressed tsRNAs in HCC were profiled.A novel tsRNA,tRNAGln-TTG derived 5′-tiRNA-Gln,is significantly downregulated,and its expression level is correlated with progression in patients.In HCC cells,5′-tiRNA-Gln overexpression impaired the proliferation,migration,and invasion in vitro and in vivo,while 5′-tiRNA-Gln knockdown yielded opposite results.5′-tiRNA-Gln exerted its function by binding eukaryotic initiation factor 4A-I(EIF4A1),which unwinds complex RNA secondary structures during translation initiation,causing the partial inhibition of translation.The suppressed downregulated proteins include ARAF,MEK1/2 and STAT3,causing the impaired signaling pathway related to HCC progression.Furthermore,based on the construction of a mutant 5′-tiRNA-Gln,the sequence of forming intramolecular G-quadruplex structure is crucial for 5′-tiRNA-Gln to strongly bind EIF4A1 and repress translation.Clinically,5′-tiRNA-Gln expression level is negatively correlated with ARAF,MEK1/2,and STAT3 in HCC tissues.Collectively,these findings reveal that 5′-tiRNA-Gln interacts with EIF4A1 to reduce related mRNA binding through the intramolecular Gquadruplex structure,and this process partially inhibits translation and HCC progression.

Biochemical Insights into the Role of Guanosine Oxidation on RNA G-Quadruplex

While 8-oxo-7,8-dihydro-2′-deoxyguanosine(dOG)on DNA G-quadruplex(G4)has been studied,the influence of 8-oxo-7,8-dihydro-guanosine(rOG)lesions on telomeric repeat-containing RNA(TERRA)G4 deserves an in-depth study.The single-strand and guanine-rich characters of TERRA make it vulnerable to form rOG lesions.Our current study demonstrated that rOG located in the internal layer and external layer of TERRA impacted the G4 stability in different ways and perturbed RNA replication,as well as base-pair strength and stability.

In vitro selection of G-rich RNA aptamers that target HIV-1 integrase

Aptamers that interact with various HIV-1 proteins,such as reverse transcriptase,Rev,Tat protein,and nuclear capsule protein,have been prepared through SELEX (systematic evolution of ligands by ex-ponential enrichment) technique. However,there are few reports about the DNA or RNA aptamers that target HIV-1 integrase. In this investigation,we selected alternative RNA aptamers specific for the HIV-1 integrase by using a different binding buffer containing 10 mmol·L-1 MgCl2 and 100 mmol·L-1 KCl. Aptamer IN1,IN2,IN3 had similar and the highest Kd values from 145 to 239 nmol·L-1. Structural studies showed that they formed similar stem-loop structure. Deletion of any stem structure resulted in diminished affinity. In addition,structure probing study with antisense DNA indicated that the stem-loop structure in the random region was critical for integrase binding. Although aptamer IN1 failed to form G-quartet structure,it might directly interact with the DDE motif of integrase,which is the virus DNA-binding site,because G-quadruplex T40214 competitively inhibited the interaction between IN1 and integrase. Together,this study generated a novel RNA aptamer IN1,which could be useful in basic research and anti-HIV drug screening.

Chemical and structural biology of nucleic acids and protein-nucleic acid complexes for novel drug discovery

Since nucleic acids(DNA and RNA) play very important roles in cells,they are molecular targets of many clinically used drugs,such as anticancer drugs and antibiotics.Because of clinical demands for treating various deadly cancers and drug-resistant strains of pathogens,there are urgent needs to develop novel therapeutic agents.Targeting nucleic acids hasn’t been the mainstream of drug discovery in the past,and the lack of 3D structural information for designing and developing drug specificity is one of the main reasons.Fortunately,many important structures of nucleic acids and their protein complexes have been determined over the past decade,which provide novel platforms for future drug design and discovery.In this review,we describe some useful nucleic acid structures,particularly their interactions with the ligands and therapeutic candidates or even drugs.We summarize important information for designing novel potent drugs and for targeting nucleic acids and protein-nucleic acid complexes to treat cancers and overcome the drug-resistant problems.