• Journal of Microbiology
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• 제41권3호
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• pp.239-247
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• 2003

The LRV1-4 capsid protein possesses an endoribonuclease activity that is responsible for the single site-specific cleavage in the 5' untranslated region (UTR) of its own viral RNA genome and the formation of a conserved stem-loop structure (stem-loop IV) in the UTR is essential for the accurate RNA cleavage by the capsid protein. To delineate the nucleotide sequences, which are essential for the correct formation of the stem-loop structure for the accurate RNA cleavage by the viral capsid protein, a wildtype minimal RNA transcript (RNA 5' 249-342) and several synthetic RNA transcripts encoding point-mutations in the stem-loop region were generated in an in vitro transcription system, and used as substrates for the RNA cleavage assay and RNase mapping studies. When the RNA 5' 249-342 transcript was subjected to RNase T1 and A mapping studies, the results showed that the predicted RNA secondary structure in the stem-loop region using FOLD analysis only existed in the presence of Mg$\^$2＋/ ions, suggesting that the metal ion stabilizes the stem-loop structure of the substrate RNA in solution. When point-mutated RNA substrates were used in the RNA cleavage assay and RNase T1 mapping study, the specific nucleotide sequences in the stem-loop region were not required for the accurate RNA cleavage by the viral capsid protein, but the formation of a stem-loop like structure in a region (nucleotides from 267 to 287) stabilized by Mg$\^$2＋/ ions was critical for the accurate RNA cleavage. The RNase T1 mapping and EMSA studies revealed that the Ca$\^$2＋/ and Mn$\^$2＋/ ions, among the reagents tested, could change the mobility of the substrate RNA 5' 249-342 on a gel similarly to that of Mg$\^$2＋/ ions, but only Ca$\^$2＋/ ions identically showed the stabilizing effect of Mg$\^$2＋/ ions on the stem-loop structure, suggesting that binding of the metal ions (Mg$\^$2＋/ or Ca$\^$2＋/) onto the RNA substrate in solution causes change and stabilization of the RNA stem-loop structure, and only the substrate RNA with a rigid stem-loop structure in the essential region can be accurately cleaved by the LRV1-4 viral capsid protein.

• Bulletin of the Korean Chemical Society
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• 제29권5호
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• pp.1063-1064
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• 2008

• Bulletin of the Korean Chemical Society
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• 제29권11호
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• pp.2227-2230
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• 2008

• 한국자기공명학회논문지
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• 제20권2호
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• pp.46-49
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• 2016

MiRNA-155, upregulated in various cancers, is one of the miRNAs that suppress apoptosis of human cancer. Thus, inhibition of the maturation of miRNA-155 could be an effective way to induce apoptotic cancer cell death. The apical stem-loop of the pre-miRNA-155 has been known as a Dicer biding site for RNA cleavage. Here, to understand the molecular basis of the tertiary interaction between pre-miRNA-155 with Dicer, we characterize the structure of the apical stem-loop of pre-miRNA-155 using NMR spectroscopy. The RNA has a stem-bulge-stem-loop-stem structure, which is consist of G-C Watson-Crick and G-U Wobble base pairs. The assignments of imino- protons were further confirmed by 2D $^{15}N-^1H$ HSQC NMR spectrum. The NMR parameters obtained in this study can be further used to investigate the tertiary interaction between pre-miRNA-155 and other biomolecules such as protein, nucleic acids, or small chemicals which might be used to control the apoptosis of cancer.

• 생명과학회지
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• 제29권6호
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• pp.653-661
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• 2019

본 연구는 siRNA 기반 치료제등의 핵산치료제 개발에 있어서 필수적인 약물의 생체내 흡수, 분포, 대사, 배설에 대한 동태의 확인을 위해 stem-loop RT-qPCR 법을 이용하여 보다 더 정확한 시험법을 확립하고자 하였다. siRNA에 특이적인 primer와 probe를 선별하여 siRNA 정량검출 시험법을 최적화하였다. siRNA 표준시료를 이용하여 최적화된 시험법을 적용하였을 때 siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과, 기울기 평균 -3.3, 결정계수 $R^2$>0.99으로 확인되어 siRNA 표준시료와 Cp 값 간의 회귀성이 매우 높아 정량 분석이 가능한 시험법임을 확인하였고, 같은 표준시료를 이용한 stem-loop RT-qPCR의 검출한계(LOD)는 10 fM, 최소정량한계(LLOQ)는 100 fM이었다. 확립된 시험법의 신뢰성을 확인하기 위해 시험자를 다르게 하고, 시험법을 3회 반복하여 각각 진행한 결과, siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과 기울기와 결정계수 $R^2$의 재현성(slope ${\pm}-3.2$, 결정계수 $R^2$>0.99)을 확인하였고, 표준 곡선으로부터 환산된 siRNA 표준시료의 회수율(recovery ${\pm}20%$)과 완건성이 우수함을 확인하였다. 확립된 stem-loop RT-qPCR을 생체내 존재하는 약물 검증에 적용할 수 있는지 확인하기 위하여 시험동물에 siRNA를 주입 후 시간별 혈액을 채취하여 확립된 시험법으로 시험을 진행하였고 약물 동태학적 분석을 통해 siRNA치료제의 혈액내의 안정성을 확인하였다. 따라서 본연구에서 개발된 stem-loop RT-qPCR 분석법은 정확성, 정밀성 및 민감도가 높은 분석법으로 핵산치료제 개발 연구의 다양한 생체시료 분석 연구에 적용할 수 있을 것으로 기대한다.

• Bulletin of the Korean Chemical Society
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• 제26권12호
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• pp.2033-2037
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• 2005

In vitro selection was used to isolate $Mg^{2+}$-dependent self-cleaving ribozymes with cis-cleavage activity from a pre-tRNA library having 40-mer random sequences attached to 5'-end of E. coli $tRNA^{Phe}$. After 8 rounds of SELEX (Systematic Evolution of Ligands by Exponential Enrichment), RNA molecules which can self-cleave at the high concentration of $Mg^{2+}$ were isolated. The selected ribozymes can carry out the self-cleavage reaction in the presence of 100 mM $Mg^{2+}$ but not in 10 mM $Mg^{2+}$. The cleavage sites of the ribozymes are located at +3 and +4 of $tRNA^{Phe}$, compared with +1 position of 5'-end cleavage site of pre-tRNA by RNase P. New RNA constructs deprived of its D stem-loop, anticodon stem-loop, variable loop and T stem-loop, respectively showed the cleavage specificity identical to a ribozyme having the intact tRNA structure. Also, the new ribozyme fused with both a ribozyme and $tRNA^{Leu}$ showed the cleavage activities at the various sites within its sequences, different from two sites of position +3 and +4 observed in the ribozyme with $tRNA^{Phe}$. Our results suggest that the selected ribozyme is not structural-specific for tRNA.

• 생명과학회지
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• 제6권4호
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• pp.234-240
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• 1996

PKR인산화효소의 억제인자로서 밝혀진 이중선RNA결합단백질 (RBF)의 RNA결합특이성을 정기영도에 의한 RNA 이동변화실험과 여과막결합도실험에 의해 측정하였다. RBF는 바이러스RNA나 stem/loop구조를 지니는 합성 RNA들에 대한 다양한 친화력을 지니는 것으로 나타났으며 충분한 GC가 포함된 11염기쌍으로 이루어진 RNA stem helix RBF가 결합하기 위한 최소한의 RNA구조로 제시되고 있다. 자연적 RNA구조에 대한 RBF의 결합은 poly(I) : poly(C)의 첨가에 의해 반전되었으며 E. coli 5S RNA경우는 효과를 거의 나타내지 않았다.

• 미생물학회지
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• 제35권3호
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• pp.211-217
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• 1999

표적 RNA 의 구조가 Tetrahymena thermophila 의 group I intron 에 의한 trans-splicing 반응에 미치는 영향을 분석하기 위해 강력한 stem-loop 형태의 안정된 구조를 갖고 있는 표적 RNA mapping 분석 방법을 이용한 결과 in vitro 뿐만 아니라 in vivo 에서도 stem 부위의 염기들에 반해 loop 부위의 염기들이 ribozyme 에 의해 잘 인지되었으며 이러한 결과는 그러한 부위들을 인지할 수 있는 ribozyme 들에 의한 trans-cleavage 그리고 trans-splicing 반응을 수행함으로써 검증하였다. 또한 이러한 trans-splicing 반응은 정확하게 일어남을 반응 산물의 염기서열 결정을 통해 확인하였다. 따라서 표적 RNA 의 구조가 in vitro 및 in vivo 에서의 ribozyme 활성에 매우 중요한 요인임을 확인하였다.

• Animal Bioscience
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• 제37권6호
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• pp.1021-1030
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• 2024

Objective: R-loops are DNA:RNA triplex hybrids, and their metabolism is tightly regulated by transcriptional regulation, DNA damage response, and chromatin structure dynamics. R-loop homeostasis is dynamically regulated and closely associated with gene transcription in mouse zygotes. However, the factors responsible for regulating these dynamic changes in the R-loops of fertilized mouse eggs have not yet been investigated. This study examined the functions of candidate factors that interact with R-loops during zygotic gene activation. Methods: In this study, we used publicly available next-generation sequencing datasets, including low-input ribosome profiling analysis and polymerase II chromatin immunoprecipitation-sequencing (ChIP-seq), to identify potential regulators of R-loop dynamics in zygotes. These datasets were downloaded, reanalyzed, and compared with mass spectrometry data to identify candidate factors involved in regulating R-loop dynamics. To validate the functions of these candidate factors, we treated mouse zygotes with chemical inhibitors using in vitro fertilization. Immunofluorescence with an anti-R-loop antibody was then performed to quantify changes in R-loop metabolism. Results: We identified DEAD-box-5 (DDX5) and histone deacetylase-2 (HDAC2) as candidates that potentially regulate R-loop metabolism in oocytes, zygotes and two-cell embryos based on change of their gene translation. Our analysis revealed that the DDX5 inhibition of activity led to decreased R-loop accumulation in pronuclei, indicating its involvement in regulating R-loop dynamics. However, the inhibition of histone deacetylase-2 activity did not significantly affect R-loop levels in pronuclei. Conclusion: These findings suggest that dynamic changes in R-loops during mouse zygote development are likely regulated by RNA helicases, particularly DDX5, in conjunction with transcriptional processes. Our study provides compelling evidence for the involvement of these factors in regulating R-loop dynamics during early embryonic development.

• Bulletin of the Korean Chemical Society
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• 제30권5호
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• pp.1193-1194
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• 2009