Development of near-infrared fluorescent probes for image-guided precision lung cancer surgery

Rho, Jiyun 고려대학교 대학원 2023 국내박사

Listeria sp. 검출용 Bioreceptor 개발

이광수 배재대학교 대학원 2010 국내석사

Listeria sp.는 최근 냉동식품과 유제품등 냉장유통 제품의 급격한 증가와 수입식품이 개방화 되고 집단 급식이 증가함에 따라 집단 식중독 사고가 증가하고 있으며 치사율이 높아 주의 깊은 관리와 예방이 요구되고 있는 현실에서 병원균에 대한 특이적 감지 및 검출법의 개발은 매우 필요하다. 따라서 본 연구는 병원균인 Listeria sp.에 대한 특이적 감지를 위하여 virulence factor인 PrfA 단백질 및 LisA peptide를 항원으로 사용하여 1차 항체를 제작하였다. PrfA 단백질의 경우 Ni-NTA resin을 이용하여 His·Tag된 분자량 23kDa 크기의 단백질을 분리·정제하였고, LisA peptide의 경우 epitope prediction을 통하여 가장 특이성이 높은 LisA 단백질의 C-PIEKKHADEIDKYIQG (17mer)를 합성하여 1차 항체를 제작하였다. 제작된 항체를 적용한 Immunosensor chip 및 Immuno-fluorescent PSQ nanoparticle을 이용하여 Listeria sp.에 특이적·신속한 감지 및 검출조건을 확립하고자 하였다. 본 연구를 통해 제작된 바이오센서는 단백질칩으로, SPR sensor chip을 이용한 비표지 방식과 형광물질를 이용한 표지방식을 적용 하였다. 비표지 방식인 SPR sensor chip은 glass surface에 얇은 금막을 형성 시키고 SAM과 dextran 층을 형성 시킨 후 제작된 항체를 이용하여 칩 표면에 약 3000Ru 값 이상을 고정화하였다. 그러나 whole cell과의 반응에서 신호가 미약하여 바이오센서칩에 적용 할 수 없었다. 따라서 기존 제작된 Listeria sp. - Unconjugated Preparation rabbit IgG polyclonal Ab를 적용하여 SPR을 수행한 결과 9.0×107CFU/ml 농도의 Listeria monocytogenes를 신속하게 검출하였다. 또한 표지방식으로 활용할 immuno-fluorescent PSQ nanoparticle은 형광물질인 rhodamine을 내포한 fluorescent PSQ nanoparticle에 항체를 고정화 시켜 바이오센서칩에 적용하였으며, SEM과 형광현미경을 이용하여 많은 광량의 발생과 특이성을 확인하였다. 본 연구를 통하여 얻어진 결과를 볼 때 항체-whole cell의 결합력을 이용한 다양한 병원균에 대한 바이오센서 적용가능성을 확인하였으며, 확립된 immunosensor chip 제조기술 및 immuno-fluorescent PSQ nanoparticle 적용 조건은 보다 특이적이고 신속한 병원균의 검출에 유용하게 활용될 것으로 사료된다. Recently, the refrigerated foodstuff like frozen food and milk products are prevalent. Also the imported food is supplying greatly and the mass-provided food is very popular. One of the pathogenic bacteria which is related with the food poisoning disease is Listeria monocytogenes. The massive food poisoning and the death rate get higher. It is necessary that the research and technology for sensing and detecting Listeria monocytogenes very specifically and rapidly should be developed and improved. It is important to develop the biosensor whih can detect the bacteria so as to prevent infection and proliferation of the pathogenic bacteria. In this study, virulence factors(PrfA, LisA) were selected as the specific proteins for the detection of Listeria monocytogenes, and then those genes were cloned and obtained the pure protein(namely, antigenic protein) through the gene cloning and overexpression system. For the strong epitope prediction of antigenic protein domain, the region where hydrophobicity is low and antigenicity is high should be likely to have high possibility producing specific antibody. C-PIEKKHADEIDKYIQG (17mer) of LisA domain where it might be exposed to the outside in the three dimensional structure were synthesized. In further stage, the synthesized peptide was combined with carrier and injected into a rabbit. After the 3 times of boosting, the serum was obtained and the antibody was purified.. Also Listeria sp. - Unconjugated Preparation rabbit IgG polyclonal ab, which is purchased from antibody production corporation, was used to make the bioreceptor. In this research, bioreceptor is the kind of protein chip, and non-mark devices using the SPR to get the signal. Into the bargain, mark devices using the in fluorescent substance in the form of PSQ nanoparticle were applied. SPR sensor chip which is non-mark devices comprised a thin gold film on the glass surface and immobilized of Ab against such as PrfA, LisA and Listeria sp. - Unconjugated Preparation rabbit IgG polyclonal Ab onto SAM, dextran layer. Applying to SPR apparatus, it was shown as the value of 3000 RU. Then, Listeria monocytogenes. 9.0×107CFU/ml could be detected. Also, the immuno-fluorescent nanoparticles bound antibody which can be utilized as a mark devices for the detection of Listeria monocytogenes were constructed through the immobilization of antibody onto the fluorescent PSQ nanoparticle comprising rhodamine as a fluorescent dye. As the results from the analyses with the formation of nanoparticle-antibody complex and with the reaction between this complex and pathogenic bacteria directly and onto the biosensor chip, defects of the photon immune sensor including irradiance and specificity were overcome. Through these researches, SPR sensor chip and immunonanoparticle that we made can be applied to various biosensor. The manufacture technology of immunosensor chip and immuno-fluorescent PSQ nanoparticle could help to make immuno-biosensor, photon-immuno sensor and fluorescent immuno-sensor. Pathogenic bacteria infect host’s body and cause illness, so using the biosensor, we need to find out them quickly and accurately if there were pathogenic bacteria to prevent illness.

Naphthalimide based fluorescent probes for various enzymes detection and cellular imaging

박선영 숙명여자대학교 대학원 2020 국내석사

Fluorescent probes can image various materials selectively that present in vivo. Since various substances are overexpressed in cancer cells, developing a fluorescent probe capable of imaging them will help distinguish cancer cells from normal cells and further aid in the diagnosis and treatment of diseases. In this paper, we designed and synthesized three fluorescent probes using naphthalimide to study the fluorescence changes of many enzymes that over-expressed in cancer cells. In the first study, the fluorescent probe was developed to detect hNQO1 (humun NAD (P) H quinone oxidoreductase type 1) by introducing coumarin and trimethyl lock quione (TLQ) into naphthalimide. TLQ acts as a reactive moiety of the hNQO1 enzyme selectively, as well as producing a PET effect that can cause fluorescence changes after the reaction. In the second study, triphenyl phosphate (TPP) was introduced to naphthalimide –TLQ conjugated compound to develop a probe that is more soluble and much more responsive to hNQO1 than the previous study. The reference probe confirmed that the activity of the probe which TPP was introduced was better. In addition, when the hNQO1-positive cell line and the hNQO1-negative cell line were compared, it was confirmed that the fluorescence was expressed in the hNQO1-positive cell line, and the fluorescence was reduced by dicoumarol. In the third study, developed a ratiometric fluorescent probe that capable of detecting tyrosinase, which is highly expressed in melanoma, a malignant skin cancer. A morpholine was introduced to detect tyrosinase in the melanosomes in melanocytes. Comparing cells that were overexpressed with tyrosinase versus cells that were not expressed with tyrosinase showed a clear fluorescence change from blue fluorescence to yellow fluorescence. Inulinvosin, which can be used as a therapeutic agent for melanoma by reducing tyrosinase, was also treated with a probe synthesized with the effect of the drug. 형광 프로브 (fluorescent probe)는 생체 내 다양한 물질들을 선택적으로 이미징 할 수 있다. 암세포 내에는 다양한 물질들이 높은 농도로 존재하기 때문에 이를 이미징 할 수 있는 형광 프로브를 개발하면 암세포와 정상 세포를 구별하고 더 나아가서 질병의 진단 및 치료에 도움이 될 것이다. 이 논문에서는 나프탈이미드 (naphthalimide) 형광체를 이용하여 세 가지 형광 프로브 구조를 디자인 및 합성하여 암세포에 많이 존재하는 효소들에 대한 형광 변화를 연구하였다. 첫 번째 연구에서는 나프탈이미드에 쿠마린과 트리메틸락퀴논 (trimethyl lock quione, TLQ)을 도입하여 생체 내 hNQO1 (humun NAD(P)H quinone oxidoreductase type 1)을 검출할 수 있는 형광 프로브를 개발하였다. TLQ는 선택적으로 hNQO1 효소에 반응하는 부분으로 작용할 뿐 아니라 PET 효과를 일으켜 반응 후에 형광 변화를 일으킬 수 있다. 두 번째 연구에서는 트리페닐포스페이트 (Triphenyl phosphate, TPP)를 도입하여 앞의 연구보다 용해도가 좋고 hNQO1에 반응성이 훨씬 더 좋은 프로브를 개발하였다. 비교 물질을 통해 TPP가 도입된 프로브의 활성이 더 좋은 것을 확인하였다. 또한 hNQO1 양성 세포주와 hNQO1 음성 세포주를 비교했을 때 hNQO1 양성 세포 주에서 형광이 많이 발현되는 것을 확인하였고, hNQO1활성을 저해시키는 물질인 디쿠마롤 (dicoumarol)을 처리하였을 때 형광이 감소하는 것을 확인하였다. 세 번째 연구는 악성 피부 암인 흑색종에 특이적으로 많이 발현되는 티로시나아제 (tyrosinase)를 감지할 수 있는 비율 계량 (ratiometric) 형광 프로브를 개발하였다. 멜라닌 세포 (Melanocyte)내의 멜라노좀 (Melanosome)에 티로시나아제를 감지할 수 있게 모르폴린 (morpholine)을 도입하였다. 티로시나아제가 과발현 된 세포와 그렇지 않은 세포를 비교하였을 때 티로시나아제가 과발현 된 세포에서 파란 형광에서 노란 형광으로의 확실한 형광 변화를 보였다. 또한 티로시나아제를 감소시킴으로써 흑색종의 치료제로 사용될 수 있는 이눌라보신 (Inulavosin)을 처리하여 약물의 효과를 확인하였다.

Synthesis and analysis of fluorescent molecules for detection of redox species and its application to biological systems

윤신아 숙명여자대학교 대학원 2024 국내박사

Development of Two-Photon Probes for Lipid Droplets Screening and NAD(P)H Quinone Oxidoreductase Activity

조명기 아주대학교 2021 국내박사

The fluorescent probes are useful in research observing various diseases and biological phenomena. Small molecule fluorescent probes have a low molecular weight, so they are easily loaded to cells and tissues. Thus, small molecule fluorescent probes can be easily applied to monitoring various biological events via confocal microscopy. There are several types of fluorescent probes, one of which is to use two-photon fluorescence. Two-photon fluorescent probes, with exceptional high transmittance originating from the use of a near-infrared excitation source, have been utilized in deep-tissue imaging which is out of scope for the applications of common one-photon fluorescent probes. In addition, two-photon microscopy utilizes relatively lower energy for excitation of photons damaging tissue samples minimally and thereby various tissue-imaging experiments with long-exposure time are feasible due to this high durability. Therefore, two-photon fluorescent probes can be applied to a variety of biomarkers and utilized in live tissues imaging. We employed two-photon probes for screening and detecting cellular organelles and enzymes. Lipid droplets (LDs) are one of the cellular organelles. LDs contain the neutral lipids such as triglycerides, cholesterol and acyl glycerol. The role of LDs is to regulate the amount of lipids in cells and supply stored lipids as a source of energy for cells. In particular, LDs rapidly increase in size and number when the endoplasmic reticulum (ER) is exposed to stress or when lipid metabolism is defective. Therefore, by tracking the size and number of LDs, it is possible to confirm the abnormal conditions or diseases. Thus, we designed a two-photon fluorescent probes for staining detecting LDs. We developed the probes that emit green and red fluorescence and observed changes in LDs under ER-stress and disease in which lipids were rapidly increased. Probes 3 and 4, which are selective for LDs, emit green and red fluorescence, respectively, and have a large stokes shift and narrow emission spectrum. Because of these characteristics, Probe 3 and 4 could perform multicolor imaging with other organelle trackers. In this study, the changes of LDs in drug-induced liver injuries were observed. Steatosis, one of the liver diseases, result in a large amount of lipids accumulated in cells due to liver toxicity, and steatosis can be caused by drugs which induce liver toxicity. Therefore, various drugs were treated in liver cells and tissues, and we monitored the liver toxicity. Based on the research results, Probe 3 and 4 can be applied to monitor of drug-induced liver toxicity. Through multicolor imaging with lysosome trackers, Probe 3 and 4 can distinguish between steatosis and phospholipidosis which is similar but different with steatosis. Thus, it is expected that Probes 3 and 4 can be useful in diverse liver disease studies and screening of various drugs. Human NAD(P)H quinone oxidoreductase type 1 (hNQO1) is an enzyme present in the cytoplasm that reduces intracellular toxic quinone derivatives, allowing cells to maintain homeostasis from oxidative stress. In particular, the expression of hNQO1 is significantly increased in cancer compared to normal. It is due to oxidative stress caused by the proliferation of cancer, especially liver and colon cancer show quite high activity of hNQO1. Therefore, it is possible to distinguish between normal and cancer by detecting the activity of hNQO1 enzyme. To detect the activity of hNQO1, we developed two ratiometric probes. The probes are reduced by hNQO1 enzyme, and fluorescence of probes were changed after response to hNQO1. The activity of the hNQO1 could be monitored by investigating the ratio of the changed fluorescence. SHC and SHC-E, developed for detecting hNQO1, are ratiometric probes, representing the difference in hNQO1 activity in normal and cancer. In addition, by applying to a biopsy sample of a patient with colorectal cancer, SHC and SHC-E could distinguish between normal and cancer. Furthermore, SHC could detect the progression stage of cancer. Therefore, SHC and SHC-E can be directly applied to the study of hNQO1 activity in normal and cancer, and can be used as a major tool for more detailed study of colorectal cancer and development of diagnostic methods.

Characterizing Photophysics of Photoconvertible-Fluorescent Protein mEos3.2 for Quantitative Fluorescence Microscopy

Sun, Mengyuan (Helen) ProQuest Dissertations & Theses Yale University 2021 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Photoconvertible fluorescent proteins (PCFPs) are widely used in super-resolution microscopy, and studies of cellular dynamics. Their photoconversion properties have enabled single-molecule localization microscopy (SMLM) by temporally separating closely-spaced molecules. However, our understanding of their photophysics is still limited, hampering their quantitative application. For example, counting fluorescently-tagged fusion proteins from the discrete localizations of individual molecules is still difficult. The red-to-green photoconvertible fluorescent protein mEos3.2 is favored by many due to its monomeric property, high brightness, photostability, compatibility with live cells, and 1:1 labeling stoichiometry. The fluorescent protein mEos3.2 is fused to the coding sequence of a protein of interest in the genome for endogenous expression or expressed exogenously and transiently in cells. Irradiation at 405 nm photoconverts mEos3.2 molecules from their native green state with an emission peak at 516 nm to their red state with an emission peak at 580 nm. Sparsely distributed photoconverted red mEos3.2 are excited at 561 nm and then localized for SMLM imaging. Understanding the factors that affect mEos3.2 photophysics can greatly strengthen its applications in imaging and quantitative measurements. However, we still do not know 1) how the behavior of mEos3.2 in live cells compares with fixed cells, and how the imaging buffer influences mEos3.2 photophysics in fixed cells, 2) how different imaging methods and laser intensities affect the behavior of mEos3.2, and 3) if there are unknown dark states of mEos3.2 that can further complicate imaging and quantitative applications of mEos3.2. In this body of work, I first reviewed the usage of photoconvertible fluorescent proteins in SMLM with a focus on its quantitative application. I discussed the significance, advantages, and challenges of counting molecules of interest tagged with mEos3.2 by SMLM. I highlighted how our limited understanding of mEos3.2 photophysics hampers its application in quantitative SMLM, thus requiring further investigation. Parts of this chapter are taken from Sun et al., 2021. In Chapter 2, I combined quantitative fluorescence microscopy and mathematical modeling to estimate the photophysical parameters of mEos3.2 in fission yeast cells. I measured the time-integrated fluorescence signal per cell, and rate constants for photoconversion and photobleaching by fitting a 3-state model of photoconversion and photobleaching to the time courses of the mEos3.2 fluorescence signal per cell measured by quantitative fluorescence microscopy. My method can be applied to study the photophysical properties of other photoactivatable fluorescent proteins and photoconvertible fluorescent proteins quantitatively, an approach complementary to conventional single-molecule experiments. This chapter is taken from Sun et al., 2021.In Chapter 3, I investigated how fixation affects the photophysical properties of mEos3.2, so that I could compare experiments conducted in live and fixed yeast cells with mEos3.2. Light fixation has been used to preserve cellular structures and eliminate movements of proteins to simplify the imaging and quantification process of quantitative SMLM. I discovered that formaldehyde fixation permeabilizes the S. pombe cells for small molecules, making the photophysical properties of mEos3.2 sensitive to the extracellular buffer conditions. To find conditions where the photophysical parameters of mEos3.2 are comparable in live and fixed yeast cells, I investigated how the pH and reducing agent in the imaging buffer affect the mEos3.2 photophysics in fixed cells. I discovered that using a buffer at pH 8.5 with 1 mM DTT to image mEos3.2 in fixed cells gave similar photophysical parameters to live cells. My results strongly suggested that formaldehyde fixation did not destroy mEos3.2 molecules but partially permeabilized the yeast cell membrane to small molecules. This chapter is taken from Sun et al., 2021.In Chapter 4, I investigated the effects of fixation and imaging buffer on mEos3.2 photophysics over a wide range of laser intensities by point-scanning and widefield microscopy, and also by SMLM. This chapter is taken from Sun et al., 2021.In Chapter 5, I alternated illumination at 405- and 561-nm to investigate the effects of 405- and 561-nm illumination separately. I discovered that 405-nm irradiation drove some of the red-state mEos3.2 molecules to enter an intermediate dark state, which can be converted back to the red fluorescent state by 561-nm illumination. I established the “positive” switching behavior (off-switching by 405-nm and on-switching by 561-nm illumination) of red mEos3.2 in addition to the previously reported “negative” switching behavior (switching off by 561-nm and switching on by 405-nm illumination), which could potentially affect counting the number of localizations of red mEos3.2 by quantitative SMLM. This chapter is taken from Sun et al., 2021.In Chapter 6, I described my ongoing progress towards developing a method to count molecules with SMLM using internal standards tagged with mEos3.2. I summarized the preliminary data on the internal calibration standards that I have tried. Further work is needed to optimize the standards and test the robustness and the reproducibility of the standards. Ultimately, this work can be applied to count the number of molecules in diffraction-limited subcellular structures with SMLM by converting the number of localizations to the number of molecules.

New fluorescent chemosensors for chiral recognition of (D)- and (L)-amino acid derivatives

김윤경 부산대학교 2007 국내석사

Chiral recognition is very important because two enantiomers consisting of chiral drugs sometimes show different biological activity. In recent years, considerable efforts have been devoted to the development of chiral fluorescent chemosensors. A general approximation to the development of chiral chemosesors is the coupling of at least two units, each one displaying a precise function such as the binding site and the signaling subunit. Anthracene is a polycyclic aromatic hydrocarbon that has been widely used as a signaling subunit for cations, anions, chiral molecules. This has been so probably due to the commercial availability of a large variety of derivatives, by the fact that its photophysical properties are well-known, and because it is highly fluorescent. Particularly, photoinduced electron transfer (PET) has been extensively studied and widely used for sensing purposes of chiral moleculars as a tool of choice in fluorescent chemosensor design. In this study, we prepared two new compounds which are expected to be used as new fluorescent chemosensors. Two new compounds containing an anthracene moiety and chiral units were tested for the recognition of various anions and (D)- and (L)- amino acid derivatives as fluorescent chiral sensors. The fluorescent chemosensors showed a highly selective fluorescence quenching effect toward the racemic amino acid derivatives.

Rhodamine based Fluorescent probe for detecting Formaldehyde

김성연 Graduate School, Yonsei University 2022 국내석사

Reactive carbonyl species (RCS) are molecules with highly reactive carbonyl groups that are generated in various groups of organisms, and an exceeding amount is known to cause various human diseases. In particular, formaldehyde (FA) is the simplest form of RCS that is spontaneously produced and maintained in steady-state concentration throughout essential biological pathways. However, an abnormally elevated FA concentration can damage DNA and proteins, leading to cancers, diabetes and neurodegenerative disorders. Therefore, it is crucial to develop accurate and effective molecular tools to understand the physiological functions and the molecular mechanism of FA in vivo systems. Many methods for fast and accurate monitoring have been studied, but most of these methods lacked sensitivity and had invasive problems that caused tissue damage. To compensate these problems, researches on fluorescent sensors are drawing attentions to achieve high sensitivity and selectivity with minimal invasiveness. Herein, new rhodamine cyclic hydrazide fluorescent probes have been developed for FA detection based on imine formation reaction. Specifically, the hydrazine derivative with an amine unit induces a reaction with FA to form an imine, which eventually opens the spirocycle of rhodamine and exhibits strong red fluorescence. The rhodmaine probes showed excellent selectivity and sensitivity to FA with fast fluorescence response in aqueous buffer media and cell imaging conditions with HeLa cells. The rhodamine cyclic hydrazide suggests potential use in biological studies as a FA detecting fluorescent probe. Reactice Carbonyl Species(RCS)라고 불리는 활성카보닐종은 다양한 유기체 그룹에서 생성되는 반응성이 높은 카르보닐기를 가진 분자로, 일정량 이상의 양은 다양한 인간 질병을 유발하는 것으로 알려져 있다. 그 중 포름알데히드 (FA)는 가장 단순한 형태의 RCS로 생물학적 경로 전반에 걸쳐 자발적으로 생성되고 일정한 농도로 유지된다. 그러나 비정상적으로 상승된 포름알데히드 농도는 DNA와 단백질을 손상시켜 암, 당뇨병 및 신경퇴행성 장애 등을 유발할 수 있기 때문에 생체내에서 포름알데히드의 생리적 기능과 분자 메커니즘을 이해하기 위해서는 정확하고 효과적인 분자 도구를 개발하는 것이 중요하다. 현재까지 빠르고 정확한 모니터링을 위한 많은 방법들이 연구되어 왔지만, 대부분의 방법은 감도가 부족하거나 조직 손상을 유발하는 침습적 문제를 불러 일으켰다. 이러한 점을 보완하기 위해, 최소의 침습성으로 높은 감도와 선택성을 제공하는 형광센서에 대한 연구가 주목받고 있다. 본 연구에서는 이민 형성 반응을 기반으로 하는 포름알데히드 검출을 위한 새로운 rhodamine cyclic hydrazide 형광화학센서를 개발하고자 하였다. 합성된 히드라진 유도체의 아민 단위는 이민 형성을 위해 포름알데히드와의 반응을 유도하고, 이는 결국 로다민의 spirocycle 구조를 열어 강한 붉은빛 형광을 띄게 된다 그 결과, 합성된 형광화학센서는 수용액과 HeLa 세포에서 포름알데히드에 대한 우수한 선택성과 민감도를 가지고 빠른 형광변화를 나타내었다. 따라서 rhodamine cylic hydrazide는 포름알데히드를 검출하는 형광화학센서로서 생물학적 연구에서의 개발 가능성을 제시한다.

유전체학에서의 다양한 응용을 위한 인공 DNA의 개발 및 연구

라비쿠마라 전북대학교 일반대학원 2023 국내박사

In here we developed two major methods to synthesize the artificial nucleic acids, that are found to have various application in chemical biology and diagnosis. At first direct arylation method was developed, that can undergo adenosine selective arylation in metal free condition, at ambient temperature with a simple electron withdrawing fluoroaryls. We tested 4 fluroaryls (X, Y, Zand 2a(Qn)) that can produce different fluorescence ranging from Blue, Green, Yellow and Red fluorescence with decent quantum yield. One more advantage that we observed of the arylated oligonucleotide are found to be microenvironment sensitive due to their restricted rotation near the vinyl group. The probe Z transfected and allowed to react inside the cells (HT1080), the fluoroaryl went smooth arylation on the adenosine moieties producing green, red fluorescence. The arylated Oligonucleotide RMAQn (form fluroaryl2a(Qn)) was best microenvironment sensitive amongst four we used it for detection of single nucleotide polymorphism. The arylated probe (RMAQn) undergoes restricted rotation up on forming duplex DNA producing bright fluorescence with large discriminating factor. The second, we developed enzymatic synthesis of the artificial DNA by incorporating the different nucleotide triphosphates (dUCN2TP, dUPyTP, dUHzDESATPand dUrkTP) that can produce different fluorescence. The dUCN2 and dUPy that are molecular rotors and are microenvironment sensitive are very useful to detect the DNA-Protein interaction. The dUPyc-mycDNA was enzymatically synthesized and utilized to monitor the c-mycgene regulation, the fluorescence monitoring clearly signifies the formation of c-mycG-quadrauplex and binding with nucelolin protein to stop the primer extension. Another nucleoside dUHzDESA that can undergo coordination with Cu2+ions to form a nucleoside dimer complex and the complex can be reversed into free nucleoside with the use of glutathione. The dUHzDESATP was enzymatically incorporated into different DNA, the DNA containing HzDESA functional was utilized for regulation of polymerase chain reaction (PCR) and Rolling circle amplification. The rolling circle amplification was sequentially switched off and on by alternative addition of Cu2+and glutathione (GHK) signifying the potential use in controlling the microorganism growth by controlling their DNA replication. Finally, we developed a highly fluorescent nucleoside (dUrk) that undergo aggregation induced emission. This triphosphate was used in rolling circle amplification on a circular template that formed by selective miRNA 24-3P mediated ligation. We found that dUrk aggregation after incorporated in to the long DNA (formed by rolling circle amplification) leading to high fluorescence than the triphosphate making it a suitable candidate to detect miRNAs in fM concentration.

In vivo mr imaging and quantification of primary and metastatic cancers using biomodal lentiviral vector encoding human ferritin and green fluorescent protein

조혜림 서울대학교 대학원 2015 국내박사

A combination of reporter genes for magnetic resonance imaging (MRI) and optical imaging can provide an additional level of noninvasive and quantitative information about biological processes occurring in deep tissues. Cellular MRI with a reporter gene offers the opportunity to track small numbers of tumor cells and to study metastatic processes in their earliest developmental stages in the target organs of interest. I developed a bimodal lentiviral vector to monitor deep tissue events using MRI to detect myc tagged human ferritin heavy chain (myc-hFTH) expression and fluorescence imaging to detect green fluorescent protein (GFP) expression. No cellular toxicity due to overexpression of myc-hFTH and GFP was observed in MTT and trypan blue exclusion assays. Iron accumulation was observed in myc-hFTH cells and tumors by Prussian blue staining and iron binding assays. First of all, for primary tumor imaging, the transgene construct was stably transfected into MCF-7 and F-98 cells. After transplantation of the cells expressing myc-hFTH and GFP into mice or rats, serial MRI and fluorescence imaging were performed with a human wrist coil on a 1.5T MR scanner and optical imaging analyzer for 4 weeks. The myc-hFTH cells and tumors had significantly lower signal intensities in T2 weighted MRI than mock-transfected controls (P ≤ 0.05). This is direct evidence that myc-hFTH expression can be visualized noninvasively with a 1.5T clinical MR scanner. Second, for the noninvasive imaging and quantification of metastatic melanoma cells in the lymph nodes (LNs) of living mice, a B16F10 murine melanoma cell line expressing hFTH and GFP was constructed to allow the detection of cells by MRI and fluorescence imaging. Stable overexpression of hFTH and GFP in B16F10 murine melanoma cells was feasible and showed no cellular toxicity. In addition, hFTH cells were detectable by 9.4T MRI in vitro and in vivo, yielding significant changes in T2* relative to control cells. In BALB/c nude mice, the presence of hFTH and GFP expressing metastatic melanoma cells in deep seated axillary LNs was demonstrated as areas of low T2* on MRI, but the same LNs were not visible by fluorescence imaging because the light was unable to penetrate the tissue. Furthermore, the metastatic volume of each LN, which was assessed by cumulative histogram analysis of the T2* MRI data, correlated well with tumor burden, which was determined by histology (r = -0.8773, p = 0.0001). This study shows that MRI and fluorescence imaging of transplanted cells or metastatic cancer cells at molecular and cellular levels can be performed simultaneously using my bimodal lentiviral vector system. In addition, this techniques can be used to monitor tumor growth, regression during cell and gene-based therapy in deep tissues.