Bioimaging of Nucleolin Aptamer-Containing 5-(N-benzylcarboxyamide)-2′-deoxyuridine More Capable of Specific Binding to Targets in Cancer Cells

Chemically modified nucleotides have been developed and applied into SELEX procedure to find a novel type of aptamers to fit with targets of interest. In this study, we directly performed chemical modification of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (called 5-BzdU) in the AS1411 aptamer, which binds to the nucleolin protein expressed in cancer cells. Forty-seven compounds of AS1411-containing Cy3-labeled 5-BzdU (called Cy3-(5-BzdU)-modified-AS1411) were synthesized by randomly substituting thymidines one to twelve in AS1411 with Cy3-labeled 5-BzdU. Both statistically quantified fluorescence measurements and confocal imaging analysis demonstrated at least three potential compounds of interest: number 12, 29 and 41 that significantly increased the targeting affinity to cancer cells but no significant activity from normal healthy cells. These results suggest that the position and number of substituents in AS1411 are critical parameters to improve the aptamer function. In this study, we demonstrated that chemical modification of the existing aptamers enhanced the binding and targeting affinity to targets of interest without additional SELEX procedures

A study of microRNAs in silico and in vivo

MicroRNAs (miRNAs) are a class of small noncoding RNA molecules that account for 1% of the genome; they are transcribed by RNA polymerase II in the nucleus, processed into a single-stranded mature miRNA of 19–25 nucleotides by the enzyme Dicer in the cytoplasm, and then destabilize or inhibit the translation of their target mRNAs in the genomes of plants and animals. About 1000 miRNAs have been identified by experimental and bioinformatic analysis since the first miRNA, lin-4, was identified in Caenorhabditis elegans in 1993. Recent evidence suggests that the production of miRNAs is signal-specific and celltype specific, and that miRNAs modulate the posttranscriptional regulation of their targets in diverse biological regulatory systems, including cell growth, differentiation and cell death. In addition, miRNAs are involved in the molecular mechanisms of diseases such as cancers and diabetes. The miRNAs involved in diseases have been a significant focus of attention in work on miRNA–mRNA molecular networks and in clinical reagents for imaging, diagnosis and the treatment of disease. Many studies have identified and described the biology of miRNAs at the molecular and cellular levels. However, we are just beginning to understand the expression of miRNAs and how they regulate their targets in the development of disease.

Molecular Beacon-Based Microrna Biosensor for Imaging EPC-Treated Cellular Therapy of Ischemia

International audienc

Noninvasive imaging of microRNA124a-mediated repression of the chromosome 14 ORF 24 gene during neurogenesis

The function of microRNAs (miRNAs) is translational repression or mRNA cleavage of target genes by binding to 3`-UTRs of target mRNA. In this study, we investigated the functions and the target genes of microRNA124a (miR124a), and imaged the miR124a-mediated repression of chromosome 14 open reading frame24 (c14orf24, unknown function) during neurogenesis, using noninvasive luciferase systems. The expression and functions of miR124a were investigated in neuronal differentiation of P19 cells (P19 is a mouse embryonic carcinoma cell line) by qRT-PCR and RT-PCR. The predicted target genes of miR124a were found by searching a bioinformatics database and confirmed by RT-PCR analysis. Remarkable repression of c14orf24 by miR124a was detected during neurogenesis, and was imaged using in vitro and in vivo luciferase systems. The expression of miR124a was highly upregulated during neuronal differentiation. Overexpression of miR124a in P19 cells resulted in a preneuronal gene expression pattern. MicroRNA124a-mediated repression of c14orf24 was successfully monitored during neuronal differentiation. Also, c14orf24 showed molecular biological characteristics as follows: dominant expression in the cytoplasm; a high level of expression in proliferating cells; and gradually decreased expression during neurogenesis. Our noninvasive luciferease system was used for monitoring the functions of miRNAs, to provide imaging information on miRNA-related neurogenesis and the miRNA-regulated molecular network in cellular metabolism and diseases.This work was supported by the Nano Bio Regenomics Project (2005-00113) and by the National R&D Program for Cancer Control of the Ministry of Health & Welfare (0820320).Kim HJ, 2008, J NUCL MED, V49, P1686, DOI 10.2967/jnumed.108.052894Ko MH, 2008, FEBS J, V275, P2605, DOI 10.1111/j.1742-4658.2008.06408.xSalic A, 2008, P NATL ACAD SCI USA, V105, P2415, DOI 10.1073/pnas.0712168105Lee JY, 2008, J NUCL MED, V49, P285, DOI 10.2967/jnumed.107.042507YU JY, 2008, EXP CELL RES, V14, P2618Makeyev EV, 2007, MOL CELL, V27, P435, DOI 10.1016/j.molcel.2007.07.015Baroukh N, 2007, J BIOL CHEM, V282, P19575, DOI 10.1074/jbc.M611841200Zhu SM, 2007, J BIOL CHEM, V282, P14328, DOI 10.1074/jbc.M611393200Visvanathan J, 2007, GENE DEV, V21, P744, DOI 10.1101/gad.1519107Cao XW, 2007, GENE DEV, V21, P531, DOI 10.1101/gad.1519207Deo M, 2006, DEV DYNAM, V235, P2538, DOI 10.1002/dvdy.20847Ottobrini L, 2006, MOL CELL ENDOCRINOL, V246, P69, DOI 10.1016/j.mce.2005.11.013SOKEN T, 2006, J PHARM SCI, V101, P267NORIO N, 2006, BIOCHEM BIOPH RES CO, V350, P1006Tannous BA, 2005, MOL THER, V11, P435, DOI 10.1016/j.ymthe.2004.10.016Smirnova L, 2005, EUR J NEUROSCI, V21, P1469, DOI 10.1111/j.1460-9568.2005.03978.xLim LP, 2005, NATURE, V433, P769, DOI 10.1038/nature03315Lewis BP, 2005, CELL, V120, P15, DOI 10.1016/j.cell.2004.12.035Doubrovin M, 2004, BIOCONJUGATE CHEM, V15, P1376, DOI 10.1021/bc0498572Ambros V, 2004, NATURE, V431, P350, DOI 10.1038/nature02871Bartel DP, 2004, CELL, V116, P281Kim J, 2004, P NATL ACAD SCI USA, V101, P360, DOI 10.1073/pnas.2333854100Lee Y, 2003, NATURE, V425, P415, DOI 10.1038/nature01957Xu PZ, 2003, CURR BIOL, V13, P790, DOI 10.1016/S0960-9822(03)00250-1Brennecke J, 2003, CELL, V113, P25Dostie J, 2003, RNA, V9, P180, DOI 10.1261/rna.2141503Hutvagner G, 2002, SCIENCE, V297, P2056Lee Y, 2002, EMBO J, V21, P4663CHALFIE M, 1981, CELL, V24, P59

In vivo imaging of functional targeting of miR-221 in papillary thyroid carcinoma

MicroRNAs (miRNAs) are small, noncoding RNA molecules that control expression of target genes. The abnormally expressed miRNAs function as oncogenes or tumor suppressors in human cancer. To evaluate the abundant gene regulation of miR-221 in papillary thyroid carcinoma (PTC), we performed microarray analysis and developed a Gaussia luciferase (Gluc) reporter system regulated by miR-221. METHODS: Total RNAs were isolated from pre-miR-221-treated normal human thyroid cells (HT-ori3) and anti-miR-221-treated papillary thyroid cells (NPA). Microarray analysis was performed with 44,000 probes. The messenger RNA levels of target genes regulated by miR-221 were evaluated using reverse-transcription polymerase chain reaction. Three types of cytomegalovirus (CMV)/Gluc_3' untranslated region (UTR) of homeobox B5 (HOXB5), which included a seed sequence of mature miR-221 in the 3' UTR of HOXB5 after the Gluc stop codon, were transfected into NPA cells, and pre-miR-221 was cotransfected with CMV/Gluc_3' UTR of HOXB5. The Gluc activities in cells were measured by luciferase assay. Mice implanted with PTC-expressing Gluc regulated by miR-221 were monitored with bioluminescence imaging for 6 d. RESULTS: Microarray analysis showed thousands of genes were directly and indirectly regulated by miR-221 and shifted the gene expression pattern of normal thyroid cells toward PTC. Of several genes downregulated more than 2-fold by miR-221, messenger RNA levels of HOXB5 were significantly downregulated by miR-221. Also, in vitro or in vivo Gluc activities using CMV/Gluc_3' UTR of HOXB5 systems were downregulated dose dependently by endogenous or exogenous miR-221. CONCLUSION: MiR-221 overexpressed in PTC drives carcinoma gene expression patterns by directly and indirectly regulating numerous genes, including HOXB5. The bioluminescence imaging system using CMV/Gluc_3' UTR of HOXB5 is a useful tool for noninvasive in vivo long-term monitoring of functional targeting of miR-221

Bioimaging of geographically adjacent proteins in a single cell by quantum dot-based fluorescent resonance energy transfer

Thousands of proteins are simultaneously involved in the maintenance of a single cancer cell. Fluorescent resonance energy transfer (FRET) is one of the most general techniques for imaging biologically interacting molecules in a cell. Here, we applied FRET to image the colocalization of two proteins that do not interact biologically (nucleolin and integrin alpha(v)beta(3)), both of which are highly expressed in the plasma membrane of cancer cells. AS1411 aptamer, which targets nucleolin, was labeled by Cy3 (Cy3-AS1411) and arginine-glycine-aspartic acid (RGD) peptide, which targets integrin alpha(v)beta(3), was conjugated with quantum dot (525 nm, Qd) Qd arginine-glycine-aspartic acid (Qd-RGD). FRET activities between Cy3-AS1411 and Qd-RGD were measured in HeLa cells, a human cervical cancer cell line. FRET phenomena between Qd and Cy3 showed good compatibility according to proximity. The fluorescence signature using Qd-RGD and Cy3-AS1411 showed that nucleolin and integrin alpha(v)beta(3) proteins were highly expressed in HeLa cells. Co-incubation of Qd-RGD and Cy3-AS1411 in a single HeLa cell demonstrated that the fluorescence overlay by FRET was quantitatively and geographically quite different from that of individual confocal images. These results suggest that Qd-based FRET analysis can provide information on geographical co-localization of proteins in naive cells, which is very important for determining the molecular and cellular functions of genes involved in cancers and other clinical diseases.This work was supported by National R&D Program for Cancer Control of Ministry of Health & Welfare (0820320), National Research Foundation of Korea (No. 20090084640 and No. 2008-03767) and a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A085136).Ko MH, 2009, SMALL, V5, P1207, DOI 10.1002/smll.200801580KIM HJ, 2008, J NUCL MED, V49, P1683Anikeeva N, 2006, P NATL ACAD SCI USA, V103, P16846, DOI 10.1073/pnas.0607771103Rossie S, 2006, BRAIN RES, V1111, P1, DOI 10.1016/j.brainres.2006.06.106Dwarakanath S, 2004, BIOCHEM BIOPH RES CO, V325, P739, DOI 10.1016/j.bbrc.2004.10.099Costes SV, 2004, BIOPHYS J, V86, P3993, DOI 10.1529/biophysi.103.038422Legrand D, 2004, EUR J BIOCHEM, V271, P303, DOI 10.1046/j.1432-1033.2003.03929.xGriffiths WJH, 2003, J HISTOCHEM CYTOCHEM, V51, P613Kerbel R, 2002, NAT REV CANCER, V2, P727, DOI 10.1038/nrc905Pecheur I, 2002, FASEB J, V16, P1266, DOI 10.1096/fj.01-0911fjeXiong JP, 2002, SCIENCE, V296, P151Bates PJ, 1999, J BIOL CHEM, V274, P26369Gordon GW, 1998, BIOPHYS J, V74, P2702GARRETT KL, 1995, DEV BIOL, V169, P596SHEU JR, 1994, PEPTIDES, V15, P1391LEITINGER N, 1993, J CELL BIOCHEM, V52, P153

A reporter gene imaging system for monitoring microRNA biogenesis

MicroRNAs (miRNAs), non-coding RNA molecules, have emerged as a part of key gene regulation, participating in a variety of biological processes such as cell development. Current research methods, including northern blot and real-time PCR analysis, have been used to quantify miRNA expression. Major disadvantages of these methods include invasive techniques, such as a tissue biopsy, and the absence of repetitive studies. In this protocol we describe a simple non-invasive imaging method for monitoring miRNAs during neurogenesis. This novel method includes the design of an miRNA reporter gene vector, cell transfection, in vitro luciferase assay and in vivo bioluminescence imaging of miRNAs. Our reporter imaging system allows for repetitive, non-invasive detection of miRNAs, illustrating the miRNA124a (miR124a)-dependent decrease of Gaussia reporter activity during neuronal differentiation. Using this method, construction of a reporter-imaging vector, in vitro and in vivo signal detection steps can be carried out in similar to 10 d.This work was supported by the Brain Research Center of the 21st Century Frontier Research Program (M103KV010016-08K2201-01610), by the National R&D Program for Cancer Control of Ministry of Health & Welfare (0820320) and by the National Research Foundation of Korea (No. 20090084640).Huang B, 2009, ACTA BIOCH BIOPH SIN, V41, P231, DOI 10.1093/abbs/gmp006Kim H, 2009, MOL IMAGING BIOL, V11, P71, DOI 10.1007/s11307-008-0188-6Ko MH, 2008, FEBS J, V275, P2605, DOI 10.1111/j.1742-4658.2008.06408.xLee JY, 2008, J NUCL MED, V49, P285, DOI 10.2967/jnumed.107.042507Cao XW, 2007, GENE DEV, V21, P531, DOI 10.1101/gad.1519207Deo M, 2006, DEV DYNAM, V235, P2538, DOI 10.1002/dvdy.20847Ottobrini L, 2006, MOL CELL ENDOCRINOL, V246, P69, DOI 10.1016/j.mce.2005.11.013Tannous BA, 2005, MOL THER, V11, P435, DOI 10.1016/j.ymthe.2004.10.016Smirnova L, 2005, EUR J NEUROSCI, V21, P1469, DOI 10.1111/j.1460-9568.2005.03978.xLim LP, 2005, NATURE, V433, P769, DOI 10.1038/nature03315Pachernik J, 2005, PHYSIOL RES, V54, P115Akita H, 2004, MOL THER, V9, P443, DOI 10.1016/j.ymthe.2004.01.005Bartel DP, 2004, CELL, V116, P281SEMPERE LF, 2004, GENOME BIOL, V5Xu PZ, 2003, CURR BIOL, V13, P790, DOI 10.1016/S0960-9822(03)00250-1Brennecke J, 2003, CELL, V113, P25Dostie J, 2003, RNA, V9, P180, DOI 10.1261/rna.2141503GOULD SJ, 1988, ANAL BIOCHEM, V175, P5, DOI 10.1016/0003-2697(88)90353-3JONES HW, 1971, OBSTET GYNECOL, V38, P945