Nucleic Acid Aptamers as Tools and Drugs: Recent Developments

Rimmele, Martina

doi:10.1002/cbic.200300648

Cited by 146 publications

(73 citation statements)

References 90 publications

(117 reference statements)

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“…[9][10][11] The high selectivity and affinity of aptamers for many different ligand targets has made them a valuable tool in therapeutics, [12][13][14] diagnostics, 15 in vivo imaging, 16 regulation of gene function, 17 proteomics and genomics research, 18,19 modulation of protein function, 20 and as sensors. 21 Determining the minimum sequence requirements for selective ligand binding to an oligonucleotide is of interest.…”

Section: Introductionmentioning

confidence: 99%

Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site A

Anderson

Mecozzi

2007

Biopolymers

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We have recently introduced a computational methodology that combines molecular dynamics (MD) simulations, free-energy calculations, and in vitro binding assays to predict the minimum RNA structural requirements for selective, high-affinity RNA binding to small-molecule ligands. Here, we show that this methodology can be applied to the conformationally flexible aminoglycoside antibiotic paromomycin. A RNA consisting of an 11-mer:10-mer duplex that contains one 16S ribosome RNA decoding A-site bound to paromomycin was simulated for 4 ns. The methodology predicts that the 11-mer:10-mer duplex binds to paromomycin with high affinity, whereas smaller RNA duplexes lose complex stability and the ability to bind paromomycin. The predicted high-affinity binding to paromomycin of the 11-mer:10-mer duplex was confirmed experimentally (EC(50) = 0.28 microM), as well as the inability of smaller complexes to bind. Our simulations show good agreement with experiment for dynamic and structural properties of the isolated A-site, including hydrogen-bonding networks and RNA structural rearrangements upon ligand binding. The results suggest that MD simulations can supplement in vitro methods as a tool for predicting minimum RNA-binding motifs for both small, rigid ligands, and large, flexible ligands when structural information is available.

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Section: Introductionmentioning

confidence: 99%

Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site A

Anderson

Mecozzi

2007

Biopolymers

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“…[7][8][9][10] Although aptamers have been very effective in vitro, one of the limitations for an in vivo application is that most of aptamers, unlike small molecules, cannot be directly taken up by cells without external assistance. [11] Several inherent factors such as nucleic acids charge and size present a potent barrier to cellular uptake. The negatively charged phosphate backbone of the nucleic acid molecule is the primary cause for its inadequate and inefficient cellular association, owing to electrostatic repulsion from the negatively charged cell surface.…”

Section: Introductionmentioning

confidence: 99%

Cell‐Specific Internalization Study of an Aptamer from Whole Cell Selection

Xiao

Shangguan

Cao

et al. 2008

Chemistry A European J

227

177

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Nucleic acid aptamers have been shown many unique applications as excellent probes in molecular recognition. However, few examples are reported which show that aptamers can be internalized inside living cells for aptamer functional studies and for targeted intracellular delivery. This is mainly due to the limited number of aptamers available for cell-specific recognition, and the lack of research on their extra- and intracellular functions. One of the major difficulties in aptamers' in vivo application is that most of aptamers, unlike small molecules, cannot be directly taken up by cells without external assistance. In this work, we have studied a newly developed and cell-specific DNA aptamer, sgc8. This aptamer has been selected through a novel cell selection process (cell-SELEX), in which whole intact cells are used as targets while another related cell line is used as a negative control. The cell-SELEX enables generation of multiple aptamers for molecular recognition of the target cells and has significant advantages in discovering cell surface binding molecules for the selected aptamers. We have studied the cellular internalization of one of the selected aptamers. Our results show that sgc8 is internalized efficiently and specifically to the lymphoblastic leukemia cells. The internalized sgc8 aptamers are located inside the endosome. Comparison studies are done with the antibody for the binding protein of sgc8, PTK7 (Human protein tyrosine kinase-7) on cell surface. We also studied the internalization kinetics of both the aptamer and the antibody for the same protein on the living cell surface. We have further evaluated the effects of sgc8 on cell viability, and no cytotoxicity is observed. This study indicates that sgc8 is a promising agent for cell-type specific intracellular delivery.

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“…This property has been exploited to create shape libraries that have yielded ligands that bind with high affinity and specificity to a variety of molecules across a broad range of size and chemical composition (29,30). A number of novel diagnostics and therapeutics have been isolated from these aptamer libraries, further indicating the significant amount of shape diversity they possess (31)(32)(33)(34)(35)(36). We have initiated studies to establish whether there is enough shape diversity in a nucleic acid aptamer library to allow recognition of each component of a complex biological mixture.…”

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confidence: 99%

Deconvolution of a Complex Target Using DNA Aptamers

Fitter¹,

James

2005

Journal of Biological Chemistry

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In vitro selection of single-stranded nucleic acid aptamers from large random sequence libraries is now a straightforward process particularly when screening with a single target molecule. These libraries contain considerable shape diversity as evident by the successful isolation of aptamers that bind with high affinity and specificity to chemically diverse targets. We propose that aptamer libraries contain sufficient shape diversity to allow deconvolution of a complex mixture of targets. Using unfractionated human plasma as our experimental model, we aim to develop methods to obtain aptamers against as many proteins as possible. To begin, it is critical that we understand how aptamer populations change with increasing rounds of in vitro selection when using complex mixtures. Our results show that sequence representation in the selected population changes dramatically with increasing rounds of selection. Certain aptamer families were apparent after only three selection rounds. Two additional cycles saw a decline in the relative abundance of these families and the emergence of yet another family that accounted for more than 60% of sequences in the pool. To overcome this population convergence, an aptamer-based target depletion method was developed, and the library screen was repeated. The previous dominant family effectively disappeared from the selected populations but was replaced by other aptamer families. Insights gained from these initial experiments are now being applied in the creation of second generation plasma protein screens and also to the analysis of other complex biological targets.

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Nucleic Acid Aptamers as Tools and Drugs: Recent Developments

Cited by 146 publications

References 90 publications

Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site A

Minimum sequence requirements for the binding of paromomycin to the rRNA decoding site A

Cell‐Specific Internalization Study of an Aptamer from Whole Cell Selection

Deconvolution of a Complex Target Using DNA Aptamers

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