• BMB Reports
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• 제47권11호
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• pp.619-624
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• 2014

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability.

• 식물조직배양학회지
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• 제27권5호
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• pp.379-385
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• 2000

tRNA and 7SL RNA based antisense vehicles were prepared by inserting conserved anti-viral and anti-viroid domains. Anti-PVS coat protein leader sequence (ACPL) and antistructural antihairpin domain of PSTVd (AHII) were inserted in tRNA cassette; anti- zing finger domain of PVS, AHII and anti hop latent viroid ribozyme were inserted in 7SL RNA gene isolated from A. thaliana. These constructs were shown to be transcribed both, in in vitro and in in vivo conditions. However, it followed from our work that closely linked position of PoIII reference genes and PoIIII antisense genes within T-DNA lead to the impairment of RNA expression in transgenic plants. To assay in vivo transcription of antisense genes, hairy root potato cultures were established using h. tumefaciens A4-24 bearing both, Ri plasmid and PoIII-promoterless plant expression vectors with antisense RNA genes. Expression of antisense RNA in transgenic potato tissues was proven by specific RT-PCR reactions.

• BMB Reports
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• 제41권8호
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• pp.568-574
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• 2008

Antisense RNA molecules are powerful tools for controlling the expression of specific genes but their use in prokaryotes has been limited by their unpredictable antisense effectiveness. Moreover, appreciation of the molecular mechanisms associated with silencing in bacteria is still restricted. Here we report our attempts to define an effective antisense strategy in E. coli, and to dissect the observed silencing process. Antisense constructs complementary to different regions of lacZ were investigated, and silencing was observed exclusively upon expression of antisense RNA hybridising the 5'UTR of lac messenger. The level of lacZ mRNA was reduced upon expression of this antisense construct, and the silencing competence was found to be closely associated with its stability. These observations may help in the design of antisense molecules directed against prokaryotic genes.

• 식물조직배양학회지
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• 제25권2호
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• pp.131-134
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• 1998

Antisense PG 유전자 형질전판 토마토로부터 자식시켜 얻은 종자를 kanamycin 내성을 이용하여 5세대까지 분리, 육성하여 antisense PG 유전자가 안정적으로 고정된 식물체를 얻었다. 이들 식물체에는 genomic DNA gel blot 분석으로 antisense PG 유전자가 안정적으로 유전되며 northern blot 분석을 통하여 antisense RNA가 발현됨을 확인하였고, 또한 antisense RNA에 의한 endogenous PG 유전자의 발현 저해를 분석하였다. Antisense PG 유전자 형질전환 성숙 토마토내 PG 효소 활성이 비형질전환 성숙 토마토에 비하여 37-65% 수준으로 저하되었다.

• BMB Reports
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• 제28권6호
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• pp.538-545
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• 1995

When DNA replication of simian virus 40 (SV40) is initiated on the replication origin, the regions containing the initiation sites of DNA primase, which participates in the transient RNA primer synthesis for formation of Okazaki fragments in the lagging strand, were chosen as the target sites of antisense RNA for studies of the inhibition of SV40 DNA replication. Four recombinant transcription vectors, pUC-PrI, pUC-PrII, pGEM-PrBS, and pGEM-PrSN, coding antisense RNA, were constructed. Four antisense RNAs (named as I, II, BS, and SN) having the size of 18, 19,58, and 123 nts, respectively, were made from the transcription vectors by in vitro transcription. And then, antisense RNA in the concentration of 2${\mu}m$ were added to COS cells transfected with pATSV-W which is a recombinant plasmid containing the SV40 origin of replication. The inhibitory extent of DNA replication was measured by DpnI resistance and was confirmed by measurement of transient RNA primer synthesis. The result shows that six combinations of antisense RNA (I, II, BS, SN, I+SN, and BS+SN) lead to the inhibition of SV40 DNA replication by up to 85%.

• 농업과학연구
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• 제34권1호
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• pp.47-52
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• 2007

유전자의 발현이 다양한 형태로 억제되는 것을 silencing이라고 한다. 유전자 발현억제 방법 중 antisense RNA는 자연 상태의 mRNA에 역상보적인 RNA 분자로서 형질전환된 세포에서 그 mRNA의 전이을 억제하는 데 이용된다. RNA분자에 대하여 상보적 염기배열을 갖는 RNA는 분자간 결합을 연결하여 RNA의 기능 발현에 억제적으로 작용한다고 생각된다. 미생물에 나타나는 유전자발현 제어기작으로서 어느 특정한 mRNA에 대하여 상보적인 RNA가 유전자 발현의 억제인자로서 작용하고 있는 예가 몇 가지 알려져 있다. 이러한 경우 antisense RNA는 mRNA 상의 전이개시영역과 상보적 배열을 하고 있고, 전이과정을 방해한다고 추정되고 있지만 작용기작의 상세한 내용은 아직 명확하지가 않다. 한편 안티센스RNA는 임의의 표적유전자에 대하여 인위적으로 제작할 수가 있기 때문에 인위적인 유전자발현제어의 한 방법으로 이용되고 있다. 특정한 유전자에 대한 antisense RNA를, 발현하는 유전자를 인위적으로 제작하여 세포내에 도입하면 표적유전자의 발현을 특이적으로 억제 제어할 수 있는 것이 기대되어, 다양한 생물체를 대상으로 하여 많은 시도가 이루어지고 있으며 몇 가지 성공적인 보고가 있다. 그 중 식물이차대사과정에 관련 유전자를 대상으로 antisense RNA 기법으로 유전자의 발현억제와 이차대사산물 생산조절에 관한 연구를 이 논문에서 조사하고 정리하였다.

• 식물조직배양학회지
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• 제22권6호
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• pp.351-355
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• 1995

국내 재배종 토마토 서광 품종으로부터 분리한 Polygalacturonase 유전자(PG2)의 3'측 1.1 kb cDNA 단편을 식물 형질전환용 운반체에 antisense 방향으로 삽입한 후 자엽을 이용하여 토마토내 도입하여 형질전환 토마토를 획득하였다. 형질전환 토마토(T$^{0}$ )를 도입시켜 그 종자를 1 mg/mL 농도의 kanamycin 함유 MS 배지에서 발아시켜 분리 집단 중에서 T$_1$9 식물체를 얻었다. T$_1$9의 Genomic Southern blot 분석 결과, antisense PG 유전자 1개가 염색체 내로 삽입되었음을 확인하였고 RNA gel blot 분석으로 endogenous PG mRNA보다 antisense PG RNA가 강하게 발현됨을 확인하였다. T$_1$9 계통 10개체의 성숙 토마토 과피조직내의 PG 효소 활성도 4~60%까지 저해되었다.

• 한국가축번식학회지
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• 제24권4호
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• pp.419-428
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• 2000

랩틴은 지방세포의 비만유전자에서 분비되는 포식인자로써 음식섭취, 에너지대사, 체중, 번식생리 및 신경호르몬 분비를 조절하는 역할을 한다. 본 연구는 antisense 비만유전자를 발현하는 형질전환 생쥐를 생산하기 위하여 실시하였다. 먼저 랩틴을 분비하는 지방세포에서 RNA 를 추출한 후 역전사 PCR을 실시하여 303 bp의 anti I과 635 bp의 anti II cDNA 들을 합성하였다. 이러한 cDNA 들을 지방세포 특이적 발현 프로모터인 aP2 프로모터와 SV40 poly(A) 사이에 역방향으로 결합하여 미세주입용 유전자를 구축하였다. 생쥐의 수정란전핵에 antisense 비만유전자를 미세 주입하여 14 마리의 형질전환 생쥐를 생산하였으며, anti I 을 지닌 4 마리의 형질전환 생쥐와 anti II를 지닌 5마리의 형질전환 생쥐계통을 확립하였다. 그리고 형질전환 생쥐의 지방세포를 추출하여 RT -PCR을 실시한 결과 antisense 비만유전자 mRNA발현을 확인하였다. 따라서, 본 연구에서 생산된 형질전환생쥐는 생체 랩틴저하에 의해 비만을 일으키는 질환모텔동물로써의 사용가능성을 나타내었다.

• Molecules and Cells
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• 제46권1호
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• pp.10-20
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• 2023

Antisense oligonucleotide (ASO) technology has become an attractive therapeutic modality for various diseases, including Mendelian disorders. ASOs can modulate the expression of a target gene by promoting mRNA degradation or changing pre-mRNA splicing, nonsense-mediated mRNA decay, or translation. Advances in medicinal chemistry and a deeper understanding of post-transcriptional mechanisms have led to the approval of several ASO drugs for diseases that had long lacked therapeutic options. For instance, an ASO drug called nusinersen became the first approved drug for spinal muscular atrophy, improving survival and the overall disease course. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Although Trikafta and other CFTR-modulation therapies benefit most CF patients, there is a significant unmet therapeutic need for a subset of CF patients. In this review, we introduce ASO therapies and their mechanisms of action, describe the opportunities and challenges for ASO therapeutics for CF, and discuss the current state and prospects of ASO therapies for CF.

• Bulletin of the Korean Chemical Society
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• 제27권5호
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• pp.657-662
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• 2006

Identification of accessible sites in targeted RNAs is a major limitation to the effectiveness of antisense oligonucleotides. A class of antisense oligodeoxynucleotides, known as the “10-23” DNA enzyme or DNAzyme, which is a small catalytic DNA, has been shown to efficiently cleave target RNA at purine-pyrimidine junctions in vitro. We have designed a strategy to identify accessible cleavage sites in the target RNA, which is hepatitis C virus nonstructural gene 3 (HCV NS3) RNA that encodes viral helicase and protease, from a pool of random DNAzyme library. A pool of DNAzymes of 58 nucleotides-length that possess randomized annealing arms, catalytic core sequence, and fixed 5'/3'-end flanking sequences was designed and screened for their ability to cleave the target RNA. The screening procedure, which includes binding of DNAzyme pool to the target RNA under inactive condition, selection and amplification of active DNAzymes, incubation of the selected DNAzymes with the target RNA, and target site identification on sequencing gels, identified 16 potential cleavage sites in the target RNA. Corresponding DNAzymes were constructed for the selected target sites and were tested for RNA-cleavage in terms of kinetics and accessibility. These selected DNAzymes were effective in cleaving the target RNA in the presence of $Mg^{2+}$. This strategy can be applicable to identify accessible sites in any target RNA for antisense oligonucleotides-based gene inactivation methods.