1.Aptamer-based molecular imaging.
Protein & Cell 2012;3(10):739-754
Molecular imaging has greatly advanced basic biology and translational medicine through visualization and quantification of single/multiple molecular events temporally and spatially in a cellular context and in living organisms. Aptamers, short single-stranded nucleic acids selected in vitro to bind a broad range of target molecules avidly and specifically, are ideal molecular recognition elements for probe development in molecular imaging. This review summarizes the current state of aptamer-based biosensor development (probe design and imaging modalities) and their application in imaging small molecules, nucleic acids and proteins mostly in a cellular context with some animal studies. The article is concluded with a brief discussion on the perspective of aptamer-based molecular imaging.
Animals
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Aptamers, Nucleotide
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genetics
;
metabolism
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Base Sequence
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Biosensing Techniques
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Humans
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Molecular Imaging
;
methods
2.DNA aptamer selection in vitro for determining ketamine by FluMag-SELEX.
Mei-Qi SUN ; Fang-Qi CAO ; Xiao-Long HU ; Yu-Rong ZHANG ; Xin-Wei LU ; Li-Bo ZENG
Journal of Forensic Medicine 2014;30(5):346-349
OBJECTIVE:
To select specific DNA aptamer for determining ketamine by FluMag-SELEX.
METHODS:
Based on magnetic beads with tosyl surface modification as solid carrier and ketamine as target, a random ssDNA library with total length of 78 bp in vitro was compounded. After 13 rounds screening, DNA cloning and sequencing were done. Primary and secondary, structures were analyzed. The affinity, specificity and Kd values of selected aptamer were measured by monitoring the fluorescence intensity.
RESULTS:
Two ssDNA aptamers (Apt#4 and Apt#8) were successfully selected with high and specific abilities to bind ketamine as target with Kd value of 0.59 and 0.66 μmol/L. The prediction of secondary structure was main stem-loop and G-tetramer. The stem was the basis of stability of aptamer's structure. And loop and G-tetramer was the key of specific binding of ketamine.
CONCLUSION
FluMag-SELEX can greatly improve the selection efficiency of the aptamer, obtain the ketamine-binding DNA aptamer, and develop a new method for rapid detection of ketamine.
Aptamers, Nucleotide/metabolism*
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DNA
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DNA, Single-Stranded/genetics*
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In Vitro Techniques
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Ketamine/metabolism*
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Oligonucleotides
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SELEX Aptamer Technique/methods*
3.Cell-ELA-based determination of binding affinity of DNA aptamer against U87-EGFRvIII cell.
Yan TAN ; Huiyu LIANG ; Xidong WU ; Yubo GAO ; Xingmei ZHANG
Chinese Journal of Biotechnology 2013;29(5):664-671
A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (K(d)) for A15 were below 100 nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (K(d)) of aptamers generated by cell-SELEX.
Aptamers, Nucleotide
;
metabolism
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Brain Neoplasms
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metabolism
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Cell Line, Tumor
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Glioma
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metabolism
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pathology
;
Humans
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Mutation
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Protein Binding
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Receptor, Epidermal Growth Factor
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genetics
;
metabolism
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SELEX Aptamer Technique
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methods
4.Advance in the study of targeting delivery system for siRNA mediated by aptamers.
Xiao-Lin WANG ; Qing-Qing WANG ; Hai-Feng SONG
Acta Pharmaceutica Sinica 2012;47(7):850-855
RNA interference (RNAi), as a new technology of gene therapy, has been used in the studies of many diseases in vitro, however, targeting delivery of small interference RNA (siRNA) is still a bottleneck for clinical therapy of siRNA agents. Aptamer is a group of oligonucleotides with high affinity and targeting, and is becoming another important means of delivery for siRNA. In this review, we summarized siRNA delivery obstacles in vivo and recent attractive developments increatively using cell-internalizing aptamers to deliver siRNAs to target cells.
Aptamers, Nucleotide
;
administration & dosage
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metabolism
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Drug Delivery Systems
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methods
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Humans
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Neoplasms
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therapy
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RNA Interference
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RNA, Small Interfering
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administration & dosage
;
metabolism
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SELEX Aptamer Technique
5.Analyses of SELEX-derived ZAP-binding RNA aptamers suggest that the binding specificity is determined by both structure and sequence of the RNA.
Zhi HUANG ; Xinlu WANG ; Guangxia GAO
Protein & Cell 2010;1(8):752-759
The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including murine leukemia virus, Sindbis virus and Ebola virus, by targeting the viral mRNAs for degradation. ZAP directly binds to the target viral mRNA and recruits the cellular RNA degradation machinery to degrade the RNA. No significant sequence similarity or obvious common motifs have been found in the so far identified target viral mRNAs. The minimum length of the target sequence is about 500 nt long. Short workable ZAP-binding RNAs should facilitate further studies on the ZAP-RNA interaction and characterization of such RNAs may provide some insights into the underlying mechanism. In this study, we used the SELEX method to isolate ZAP-binding RNA aptamers. After 21 rounds of selection, ZAP-binding aptamers were isolated. Sequence analysis revealed that they are G-rich RNAs with predicted stem-loop structures containing conserved "GGGUGG" and "GAGGG" motifs in the loop region. Insertion of the aptamer sequence into a luciferase reporter failed to render the reporter sensitive to ZAP. However, overexpression of the aptamers modestly but significantly reduced ZAP's antiviral activity. Substitution of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity, suggesting that the RNA sequence is important for specific interaction between ZAP and the target RNA. The aptamers identified in this report should provide useful tools to further investigate the details of the interaction between ZAP and the target RNAs.
Aptamers, Nucleotide
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chemistry
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genetics
;
metabolism
;
Base Sequence
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Carrier Proteins
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antagonists & inhibitors
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metabolism
;
Genes, Reporter
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HEK293 Cells
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Humans
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Leukemia Virus, Murine
;
genetics
;
Molecular Sequence Data
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Nucleic Acid Conformation
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Protein Binding
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RNA
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chemistry
;
metabolism
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RNA, Viral
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genetics
;
Response Elements
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SELEX Aptamer Technique
6.Screening and characterization of DNA aptamers with hTNF-alpha binding and neutralizing activity.
Ke-Tai GUO ; Xin-Rui YAN ; Guo-Jin HUANG ; Chun-Xiao XU ; Ying-Shuang CHAI ; Zhi-Qing ZHANG
Chinese Journal of Biotechnology 2003;19(6):730-733
Human tumor necrosis factor alpha (hTNF-alpha) is one of the most important inflammatory cytokines that acts as a mediator in inflammatory and immune response and plays a key role in host defense against infection. The over expression of hTNF-alpha is associated with serious consequences, such as shock, hypotension, thrombus, septicemia and even death. It has been implicated in many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, chronic heart failure and septic shock. Inhibiting the bio-activity of hTNF-alpha is one of the strategy for the treatment of these diseases. Compared with traditional recombinant protein drugs, small molecule drugs have many advantages, such as high affinity, low immunogenecity and low cost. Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the selection of oligonucleotides that bind with high affinity and specificity to target proteins. Such oligonucleotides are called aptamers, and are potential therapeutics for blocking the activity of pathologically relevant proteins. To obtain oligonucleotide aptamers specifically binding to TNF, a 40nt random DNA combinatorial library flanked by 31nt fixed sequences was chemically synthesized. The random library was amplified with PCR and subjected to selection by SELEX protocol against hTNFalpha. After incubation of the library with hTNFalpha, the mixture was blotted onto Immobilon-NC transfer membrane. The no-specific binding was washed away and the hTNFa binding aptamers were eluted and detached from the target protein. The eluted oligo nucleotides were amplified with PCR and served as the DNA library for the next round selection. After 12 rounds of such selection, the selected aptamers were cloned to pGEM-T vector. Positive clones were identified by restriction enzyme digestion and DNA sequencing. Oligo DNA were synthesized according to the sequence data and tested for their activities. Binding activity of the aptamers to hTNFalpha were detected by ELISA and dot blot with biotin-streptavidin-horseradish peroxidase system. Mouse L929 cells were used to test the anti-hTNFa activity of the DNA aptamers. The aptamers were incubated with hTNFalpha and added to the L929 cells. The results were read under microscope and with MTT staining. It was shown that these DNA aptamers bound to hTNFalpha with high affinity, and can inhibit the cytotoxicity of hTNFalpha on cell culture. The affinity of these aptamers are different and may related to their structure. These ssDNA aptamers are potential for the treatment and diagnosis of hTNFalpha related diseases.
Animals
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Aptamers, Nucleotide
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chemical synthesis
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chemistry
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genetics
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pharmacology
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Biotin
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chemistry
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Cell Line
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Cell Survival
;
drug effects
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Enzyme-Linked Immunosorbent Assay
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Gene Library
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Humans
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Mice
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Protein Binding
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SELEX Aptamer Technique
;
methods
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Tumor Necrosis Factor-alpha
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antagonists & inhibitors
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metabolism
;
toxicity
7.Screening and structure analysis of nucleic acid aptamers binding to surface of CD33(+)/CD34(+) cells from patients with acute myeloid leukemia subtype M₂.
Shu-Qin ZHANG ; Guang-Ping WANG ; Ping ZHU ; Jia-Jia LIANG ; Ya-Jing XU ; Min-Yuan PENG ; Yan CHEN ; San-Qin TAN ; Fang-Ping CHEN
Journal of Experimental Hematology 2011;19(3):561-565
A little is known about the specific marker on the surface of acute leukemia cells, leading to the lack of the specific diagnosis method for acute leukemia. Therefore, in this study, cell-systematic evolution of ligands by exponential enrichment (cSELEX) was performed to screen the aptamers binding to CD33(+)/CD34(+) cells from the patients with acute myeloblastic leukemia (AML) of M(2) subtype (AML-M₂) so as to provide the basis for finding the specific marker on the surface of AML-M(2) CD33(+)/CD34(+) cells. Firstly, AML-M₂ CD33(+)/CD34(+) cells were sorted and used as targeted cells, and normal CD33(+)/CD34(+)cells were used as counter-targeted cells; the aptamers binding to CD33(+)/CD34(+) cells from patients with AML-M₂ were screened from the single strand deoxyribonucleic acid (ssDNA) library by cSELEX. Subsequently, each aptamer structure was analyzed after cloning and sequencing. The results indicated that after 13 round of screenings, the enrichment of aptamers in the ssDNA library was ranged from 0.7% to 52.9%, and reached steady state at 13th round screening. Sequence analysis for 30 aptamers showed that most of the aptamers born one of the three conserved sequences of CCCCT, CTCTC, and CTCAC. Secondary structure analysis indicated that three different secondary structures existed in these aptamers. It is concluded that the aptamers binding to the AML-M(2) CD33(+)/CD34(+) cells are successfully screened, which lay the basis for further looking for the specific marker on the surface of AML-M₂ CD33(+)/CD34(+) cells, and the molecular diagnosis of the AML-M₂ leukemia.
Antigens, CD
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genetics
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immunology
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Antigens, CD34
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genetics
;
immunology
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Antigens, Differentiation, Myelomonocytic
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genetics
;
immunology
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Aptamers, Nucleotide
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metabolism
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Biomarkers
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Flow Cytometry
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Humans
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Immunophenotyping
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Leukemia, Myeloid, Acute
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genetics
;
immunology
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Nucleic Acid Conformation
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SELEX Aptamer Technique
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Sialic Acid Binding Ig-like Lectin 3
8.Recent progress of the aptamer-based antiviral drugs.
Yao ZHU ; Ying-Hui LÜ ; Hui-Yong YANG ; Jun-Sheng LIN ; Qi-Zhao WANG
Acta Pharmaceutica Sinica 2013;48(4):447-456
Aptamers are capable of binding a wide range of biomolecular targets with high affinity and specificity. It has been widely developed for diagnostic and therapeutic purposes. Because of unique three dimensional structures and cell-membrane penetration, aptamers inhibit virus infection not only through binding specific target, such as the viral envelope, genomic site, enzyme, or other viral components, but also can be connected to each other or with siRNA jointly achieve antiviral activity. Taking human immunodeficiency virus and hepatitis C virus as examples, this paper reviewed the effects and mechanisms of aptamers on disturbing viral infection and replication steps. It may provide an insight to the development of aptamer-based new antiviral drugs.
Antiviral Agents
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pharmacology
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Aptamers, Nucleotide
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pharmacology
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therapeutic use
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Genome, Viral
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drug effects
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HIV
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drug effects
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HIV Reverse Transcriptase
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metabolism
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Hepacivirus
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drug effects
;
genetics
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Humans
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Macular Degeneration
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drug therapy
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Neoplasms
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drug therapy
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Oligodeoxyribonucleotides
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therapeutic use
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RNA, Small Interfering
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pharmacology
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SELEX Aptamer Technique
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Viral Envelope Proteins
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metabolism
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Virus Replication
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drug effects
9.In vitro selection of single strand deoxyribonucleic acid aptamers binding to cells from patients with acute myeloblastic leukemia.
Ping ZHU ; Guangping WANG ; Shuqin ZHANG ; Yajing XU ; Minyuan PENG ; Hui YIN ; Yan CHEN ; Sanqin TAN ; Fangping CHEN
Journal of Central South University(Medical Sciences) 2012;37(8):771-776
OBJECTIVE:
To screen aptamers binding CD33+/CD34- cells from patients with acute myeloblastic leukemia M2 subtype (AML-M2).
METHODS:
CD33+/CD34- cells from patients with AML-M2 were taken as targeted cells, CD33+/ CD34- cells from normal people were taken as anti-selecting cells, and aptamers in the single strand deoxyribonucleic acid (ssDNA) library were then selected repeatedly by cell-systematic evolution of ligands by exponential enrichment (C-SELEX) technology, and amplified by polymerase chain reaction (PCR) to generate sub-ssDNA library. During the experiment, PCR amplification with fluorescently labeled primer and flow cytometry were performed to analyze the aptamers'enrichment of sub-library, and the final round product of the sub-ssDNA library was cloned. After the sequencing, the primary and secondary structures of the aptamers were analyzed.
RESULTS:
Electrophoresis indicated that the product of PCR amplification for each round subssDNA library was able to see a clear DNA band in the agarose gel. After 13 rounds of screening, the fluorescence intensity of the sub-ssDNA library binding the cells ranged from 2.14% to 51.12%, reaching a steady state at the 13th round. A total of 30 clones were selected and sequenced, 22 of which contained 1 of the 4 conserved sequences of AAGTA, TATCT, AGATG and AAATT in their primary structure, but the remained eight aptamers contained none of the conserved sequence. Secondary structure analysis indicated that four stem-loops and loop simulation convex structures existed in the aptamers.
CONCLUSION
C-SELEX technology can be used to screen the aptamers binding primary cells from patients with leukemia. The aptamers selected from the CD33+/CD34- cells from the patients of AML-M2 subtype might be used for the diagnosis and treatment for leukemia.
Adolescent
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Adult
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Antigens, CD34
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genetics
;
immunology
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Antigens, Differentiation, Myelomonocytic
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genetics
;
immunology
;
Aptamers, Nucleotide
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genetics
;
metabolism
;
DNA, Single-Stranded
;
genetics
;
Female
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Humans
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Leukemia, Myeloid, Acute
;
genetics
;
immunology
;
Male
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Middle Aged
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SELEX Aptamer Technique
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Sialic Acid Binding Ig-like Lectin 3
;
genetics
;
immunology
;
Young Adult
10.Cotinine-conjugated aptamer/anti-cotinine antibody complexes as a novel affinity unit for use in biological assays.
Sunyoung PARK ; Dobin HWANG ; Junho CHUNG
Experimental & Molecular Medicine 2012;44(9):554-561
Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.
Animals
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Antibodies, Anti-Idiotypic/immunology/metabolism
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*Aptamers, Nucleotide/chemistry/immunology
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Biological Assay
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*Cotinine/administration & dosage/chemistry
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Flow Cytometry
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Hep G2 Cells
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Humans
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Mice
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NIH 3T3 Cells
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Phosphoproteins/*chemistry/immunology
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Protein Binding
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RNA-Binding Proteins/*chemistry/immunology
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*Vascular Endothelial Growth Factor A/chemistry/immunology