Monovalent cations effect on the binding properties of G-quadruplex; When TMPyP bound to 3G tetrads

이영애,강미숙 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0

We present the monovalent cations effect on the binding properties of G-quadruplex when TMPyP bound to multiple planar GGG tetrads. Monovalent cations like potassium and sodium have been shown to act as a stabilizing factor for the G-quadruplex structural formation. Once G-quadruplex formed by monocations, some small molecule ligands can also effect on the stability of G-quadruplex-binding molecule complexes. On the basis of the melting assay for G-quadruplex-TMPyP in K+/Na+ buffered solutions at a low ratio (R=0.2) is closely resemble to G-q alone. Which suggests that the presence of small amount of TMPyP has a minimal effect on the thermal stability of quadruplex. As a results of the job plots for G-quadruplex with TMPyP in K+ buffer, major inflection point for quadruplex-TMPyP complexes, at x = 0.70 was observed. Based on the various spectroscopic results, different monovalent cations can take an effect on the binding properties of the G-quadruplex-Porphyrin complexes.

Behavior of the Guanine Base in G-quadruplexes Probed by the Fluorescent Guanine Analog, 6-Methyl Isoxanthopterin

한지훈,Nataraj Chitrapriya,이현석,이영애,김석규,정맹준 대한화학회 2017 Bulletin of the Korean Chemical Society Vol.38 No.2

In this study, circular dichroism (CD) spectrum and fluorescence techniques were used to examine the dynamic properties and microenvironment of the guanine base (G) at the central loop and at the middle of the G-stem of the G-quadruplex formed from the G3T2G3TGTG3T2G3 sequence (G-quadruplex 1), in which the G base at the 10th and 13th position were replaced with a fluorescent G analog, 6-methyl isoxanthopterin (6MI) (G-quadruplex 2 and 3, respectively). For all G-quadruplexes, the CD spectrum revealed a positive band at 263 nm and a shoulder at 298 nm, and the thermal melting profiles were the sum of at least two sigmoidal curves. These observations indicated the presence of two conformers in the G-quadruplex. The fluorescence intensity of G-quadruplex 2 was greater than 3, as expected from the extent of stacking interaction, which is larger in the G(6MI)G sequence than the T(6MI)T sequence. The efficiency of fluorescence quenching by the polar acrylamide quencher and negatively charged I− quencher were larger for G-quadruplex 3, suggesting that 6MI in the G(6MI)G stem is exposed more to the aqueous environment compared to that in the T(6MI)T central loop. In the latter case, 6MI may direct to the center of the top G-quartet layer. The possibility of hydrogen bond formation between the carbonyl group of 6MI and the acrylamide of the G-quadruplex 3 was proposed.

Antiviral effects of G-quadruplex binding ligands against human herpesviruses

한지호(Ji Ho Han),송문정(Moon Jung Song) 고려대학교 생명자원연구소 2022 생명자원연구 Vol.30 No.-

G-quadruplexes(G4s)는 복합체를 형성하는 구아닌이 풍부한 평면 모양의 tetrad 형성의 스택으로 구성된 고전적인 이차 핵산 구조이다. G4는 주요 세포 과정의 전사, 번역, 텔로미어 유지, 후성유전적 조절, 복제 및 재조합에서 다양한 중요한 역할에 연루되어 있다. G-quadruplexes는 종양학에서 중요한 구조로 처음 발견되었지만 지난 10년 동안 다양한 바이러스와의 관련성이 더욱 분명해 지고 있다. 인간 허피스바이러스는 허피스바이러스과(Family)에 속하는 DNA 바이러스이며 숙주에서 용해성 및 잠복성 감염이라는 두 가지 유형의 감염 생활사를 갖는 것이 특징적이다. 잠복기 동안 바이러스는 평생 잠복 상태를 유지하고 간헐적으로 재활성화되어 바이러스가 숙주내, 숙주간 전파할 수 있도록 하고, 한편 숙주에서 질병 등의 문제를 야기할 수 있다. 최근에 바이러스 게놈에서 G4s의 역할과 G4에 결합하는 G4 리간드의 잠재적인 항바이러스 효능에 관한 보고가 증가하고 있다. 많은 연구 결과들이 바이러스 G4s가 바이러스 생활 주기에서 중요한 역할을 하고, G4 리간드의 치료가 용해성 및 잠복성 감염 모두에서 항바이러스 활성을 나타낼 수 있다는 것을 시사한다. 이 리뷰에서는 허피스바이러스 게놈에서 G4s의 중요성과 이러한 기전을 연구하는데 사용되는 강력한 G4 결합 리간드를 함께 소개하고, 마지막으로 각 G4 리간드의 고유한 기능적 특성을 설명하고자 한다. G-quadruplexes (G4s) are noncanonical secondary nucleic acid structures constituted by stacking of guanine rich planar shaped tetrad formations that form a complex. G4s are implicated for various important roles in key cellular processes transcription, translation, telomere maintenance, epigenetic regulation, replication, and recombination. G-quadruplexes were first discovered as important structures in oncology, but for the past decade its relevance in viruses is becoming more evident. Human herpesviruses are DNA viruses of the Herpesviridae family and are unique in characteristic with two types of infection which can be distinguished by lytic and latency establishment in the host. During latency the virus maintains lifelong dormancy and intermittently undergoes reactivation, causing the host medical problems. Recently there are increasing number of reports regarding role of G4s in viral genomes and the potential antiviral efficacy of G4 ligands, including G4s in latency. Many results suggest viral G4s play significant roles in the virus life cycle and treatment of G4 ligands exhibit antiviral activities in both lytic and latent infections. In this review, the importance of G4s in herpesvirus genomes will be introduced with the potent G4 ligands used to study these mechanisms and finally explain the distinct functional properties of each G4 ligands.

Induced another G-quadruplex structure by heavy metal ions: sensing for sensitive and selective pb²⁺ ions.

김래영,강미숙 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.1

Deoxyribose nucleic acids with the consecutive number of guanine can form a G-quadruplex structure in the presence of monovalent ions. G-quartets, consisting of four guanines connected by a hoogsteen hydrogen bond, are stacked to form a G-quadruplex. The representative DNA sequence of forming G-quadruplex is known that TBA, HTG, ARGO100, PS2.M, T2. We investigated the structural conversion by heavy metal ion using these sequences. Using Circular dichroism, G-quadrupelx structure for antiparallel structure has shown that negative band at 260nm, positive band at 290nm in CD Spectra. For parallel structure, it shown that positive band at 260nm, positive band at 290nm. However, the G-quadruplex Induced by pb²⁺ ions convert to another structure, having a negative band around 275~290nm and shifted from positive band 290nm to 315nm. We will investigate that it can detect lead in vivo through G-quadruplex structural conversion.

Fluorometric detection of influenza virus RNA by PCR-coupled rolling circle amplification generating G-quadruplex

Kim, Dong-Min,Seo, Jina,Jun, Bong-Hyun,Kim, Dong Ho,Jeong, Woong,Hwang, Sang-Hyun,Kim, Dong-Eun Elsevier 2017 Sensors and actuators. B, Chemical Vol.251 No.-

<P><B>Abstract</B></P> <P>We developed a label-free fluorometric system based on double amplification of target nucleic acid by rolling circle amplification generating G-quadruplex (GQ-RCA) for sensitive detection of influenza virus RNA. To detect viral RNA with high sensitivity and selectivity, double amplification system was employed through reverse transcription PCR (RT-PCR) coupled in tandem with GQ-RCA. Single-stranded amplified viral DNA (ssDNA) was generated by RT-PCR of samples containing influenza virus RNA. The phosphate-primed DNA strand in amplicon DNA was subsequently degraded by lambda exonuclease. Viral ssDNA formed a ternary RCA initiation complex by annealing to both a partial hairpin primer and a dumbbell padlock DNA template. A long stretch of ssDNA containing repeated copies of the G-quadruplex sequence was generated by RCA at room temperature. Sensitive detection of amplified target nucleic acid was then accomplished by monitoring fluorogenic interactions between Thioflavin T (ThT) and RCA-responsive G-quadruplexes. Differences in fluorescence intensity between target and non-target viral RNA were evident under UV illumination. Selective fluorescence staining of RCA-responsive G-quadruplexes enabled influenza virus RNA detection at concentrations as low as 4.9aM with a linear detection range between 450aM and 450fM. The RT-PCR-coupled GQ-RCA system for influenza virus genome detection exhibited very high sensitivity and allowed convenient multiplexed virus detection within 2.5h. Thus, a combination of RT-PCR-coupled GQ-RCA and ThT fluorescence staining can be a sensitive and accurate method for detecting RNA molecules of influenza viruses as well as those of other viruses.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A fluorometric assay by rolling circle amplification (RCA) generating G-quadruplex. </LI> <LI> Reverse transcription PCR coupled in tandem with RCA for double amplification. </LI> <LI> Selective fluorescence staining of RCA-responsive G-quadruplexes with Thioflavin T. </LI> <LI> Influenza virus RNA detection with a detection limit of 4.9aM. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

2-아미노퓨린을 포함하는 Split G-quadruplex를 이용한 특정 DNA 서열 검출

우지수,황성현,권우영,김석준,박기수 한국공업화학회 2019 한국공업화학회 연구논문 초록집 Vol.2019 No.1

최초로 형광 염기 유사체와 split-G-quadruplex 구조를 함께 이용하여 특정 DNA를 검출하는 간단한 기술을 개발했다. 형광 염기 유사체인 2-아미노 퓨린(2-Aminopurine;2-AP)를 포함하는 split G-quadruplex를 신호 물질로 사용하는 특정 DNA 서열 검출 방법이다. 이 방법에서 2-아미노퓨린을 포함하는 G-quadruplex는 두 부분으로 분할되고 타겟에 특이적인 overhang sequence에 연결된다. 분리된 G-quadruplex 서열은 상보적인 타겟 DNA가 있을 때만 온전한 G-quadruplex 구조를 형성하며 그 결과, 2-아미노퓨린 형광단의 형광 세기가 증가한다. 이 기술을 이용하여 매우 선택적으로 타겟 DNA를 검출했으며, 또한 인간 혈청에서 타겟 DNA를 분석함으로써 실용성이 입증되었다.

Label/Quencher-Free Detection of Exon Deletion Mutation in Epidermal Growth Factor Receptor Gene Using G-Quadruplex-Inducing DNA Probe

( Hyo Ryoung Kim ),( Il Joon Lee ),( Dong-eun Kim ) 한국미생물 · 생명공학회 2017 Journal of microbiology and biotechnology Vol.27 No.1

Detection of exon 19 deletion mutation in the epidermal growth factor receptor (EGFR) gene, which results in increased and sustained phosphorylation of EGFR, is important for diagnosis and treatment guidelines in non-small-cell lung cancer. Here, we have developed a simple and convenient detection system using the interaction between G-quadruplex and fluorophore thioflavin T (ThT) for discriminating EGFR exon 19 deletion mutant DNA from wild type without a label and quencher. In the presence of exon 19 deletion mutant DNA, the probe DNAs annealed to the target sequences were transformed into G-quadruplex structure. Subsequent intercalation of ThT into the G-quadruplex resulted in a light-up fluorescence signal, which reflects the amount of mutant DNA. Due to stark differences in fluorescence intensity between mutant and wild-type DNA, we suggest that the induced G-quadruplex structure in the probe DNA can report the presence of cancer-causing deletion mutant DNAs with high sensitivity.

NMR Investigation of the Interaction between the RecQ C-Terminal Domain of Human Bloom Syndrome Protein and G-Quadruplex DNA from the Human c-Myc Promoter

Lee, Sungjin,Lee, Ae-Ree,Ryu, Kyoung-Seok,Lee, Joon-Hwa,Park, Chin-Ju Academic Press 2019 Journal of molecular biology Vol.431 No.4

<P><B>Abstract</B></P> <P>Bloom syndrome protein (BLM) is one of five human RecQ helicases that participate in DNA metabolism. RecQ C-terminal (RQC) domain is the main DNA binding module of BLM and specifically recognizes G-quadruplex (G4) DNA structures. Because G4 processing by BLM is essential for regulating replication and transcription, both G4 and BLM are considered as potential targets for anticancer therapy. Although several studies have revealed the detailed mechanism of G4 unwinding by BLM, the initial recognition of the G4 structure by the RQC domain is unclear. Here, we investigated the interaction between BLM RQC and the G4 DNA from the c-Myc promoter by NMR spectroscopy. While the signals broadened upon reciprocal titrations, the β-wing of RQC had significant chemical shift perturbations and experienced millisecond timescale dynamics upon G4 binding. A point mutation in the β-wing (N1164A) reduced G4 binding affinity. Our hydrogen–deuterium exchange data indicate that imino protons of G4 were exchanged with deuterium much faster in the presence of RQC. We suggest that RQC binds to G4 by using the β-wing as a separating pin to destabilize the G4. By providing information about the RQC–G4 interaction, our study yields insight into potential strategies for preventing G4 processing by BLM.</P> <P><B>Highlights</B></P> <P> <UL> <LI> RecQ C-terminal (RQC) domain of Bloom syndrome protein (BLM) binds to the G-quadruplex with micromolar <I>K</I> <SUB>d</SUB>. </LI> <LI> β-wing and α2 region of the RQC domain play an important role in the G-quadruplex interaction. </LI> <LI> BLM RQC domain destabilizes G-quadruplex without the helicase domain of the protein. </LI> <LI> Our results contribute to the understanding of the initial G4 recognition process of BLM. </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Ru-complex와 이중나선, 사중나선 DNA와의 상호작용 연구

이영애 한국공업화학회 2016 한국공업화학회 연구논문 초록집 Vol.2016 No.0

Interactions of the ruthenium complexes with various nucleic acids have been widely investigated for their potential biological applications. This type of studies recently expanded for the G-quadruplexes and only a few reports started to appear. In this study, interaction of a set of enantiomer namely, Δ-, Λ-[Ru(phen)2DPPZ](2+) with human telomeric DNA, 5´-G2T2G2TGTG2T2G2-3´(15-mer), which forms a G-quadruplex in the presence of potassium ion was investigated by various spectroscopic techniques. The well-known enhancement of luminescence intensity (the 'light-switch' effect) was also observed for the [Ru(phen)2DPPZ](2+) when form an adduct with the G-quadruplex. The luminescence of the G-quadruplex bound Ru(II) complex can be quenched by external quencher. The [Ru(phen)2DPPZ](2+) complex bind at the lateral loop or under the diagonal loop and the other bind the side of the G-quadruplex.

Base sequence effect on the binding properties of G-quadruplex and Porphyrin complexes

이영애,강미숙 한국공업화학회 2018 한국공업화학회 연구논문 초록집 Vol.2018 No.0

We present the base sequence effect on the binding properties of G-quadruplex and porphyrin complexes. Change of base composition in loop or at termini of sequences have a significant effect on the stability of final structure. These resulting formations can also effect on the binding properties between G-quadruplex and porphyrin. According to results, once an extra T-base (-TTT-, S2) was placed on loop in sequence 1(-TT-, S1), Their CD spectra was quite different to each other. In contrast, when A-base (5'-A…-TTT-, S3) was added at termini of 5'-side of the sequence 2(-TTT-, S2), there is not induced any structural changes against to S2. On the melting profiles, whether extra base is placed on loop or at termini of 5'-side, less stable than the sequence that without extra base. On the basis of the various spectroscopic results, base composition can effect on the binding properties of porphyrinquadruplex complexes including their stabilities.