Oligonucleotide inhibitors of human thrombin that bind distinct epitopes

Tasset, Diane M.; Kubik, Mark F.; Steiner, Walter W.

doi:10.1006/jmbi.1997.1275

Cited by 791 publications

(820 citation statements)

References 31 publications

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“…To demonstrate the approach, we designed a 50nt ssDNA molecule, which was comprised of an aptamer domain, a poly[A] 15 linker, and a short 20nt primer ( Figure 1B). The 15nt thrombin aptamer was chosen for this study as it has been extensively characterized and binds its target with reasonable affinity (K d = ~75nM) (36)(37)(38) The primer sequence is suitable for RCA amplification, and in conjunction with the recognition domain can prime the PCR reaction. This aptamer-primer hybrid construct effectively binds to the thrombin target protein and can be directly amplified via both PCR and RCA processes.…”

Section: Introductionmentioning

confidence: 99%

Protein detection via direct enzymatic amplification of short DNA aptamers

Fischer

Tarasow

Tok

2008

Analytical Biochemistry

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Aptamers are single-stranded nucleic acids that fold into defined tertiary structures to bind target molecules with high specificities and affinities. DNA aptamers have garnered much interest as recognition elements for biodetection and diagnostic applications due to their small size, ease of discovery and synthesis, and chemical and thermal stability. Herein, we describe the design and application of a short DNA molecule capable of both protein target binding and amplifiable bioreadout processes. As both recognition and readout capabilities are incorporated into a single DNA molecule, tedious conjugation procedures required for protein-DNA hybrids can be omitted. The DNA aptamer is designed to be amplified directly by either the polymerase chain reaction (PCR) or rolling circle amplification (RCA) processes, taking advantage of real-time amplification monitoring techniques for target detection. A combination of both RCA and PCR provides a wide protein target dynamic range (1 μM to 10 pM).

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Section: Introductionmentioning

confidence: 99%

Protein detection via direct enzymatic amplification of short DNA aptamers

Fischer

Tarasow

Tok

2008

Analytical Biochemistry

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“…C15T is a distant site of TA29 from the associated thrombin in the protein-aptamer complex and has five G bases surrounded within its 4 nt region. 20,21 However, both the 3′-end and 5′-end of the TA29 only have one G base within their 4 nt regions. Among the three TA29 aptamers, only C15T-TMR-TA29 displays a significant reduction in FA (Δr = −0.081) in the presence of human α-thrombin with a molar ratio of 1:1 ( Figure 1).…”

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confidence: 99%

Fluorescence Anisotropy Reduction of Allosteric Aptamer for Sensitive and Specific Protein Signaling

Zhang

Zhao

et al. 2012

Anal. Chem.

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Real time protein signaling in a complex medium may provide a promising way for high-throughput protein analysis, but it is largely unmet due to the challenge of signal transduction and the interferences of nonspecific binding and high background. Our recent work indicates that a fluorescent aptamer can display a protein bindinginduced reduction of fluorescence anisotropy (FA) (Zhang, D.; Lu, M.; Wang, H. J. Am. Chem. Soc. 2011, 133, 9188−9191), which is exclusively different from a traditionally simplified concept hinting a molecular size increase-induced FA increase. Inspired by this unexpected observation, we describe a novel FA reduction approach for protein signaling. The feasibility of this approach is demonstrated through the assays of a blood protein human α-thrombin and an oncoprotein human platelet-derived growth factor B-chain (PDGF-BB) using two screened fluorescent aptamers, respectively. By the developed FA reduction method, the spiked human α-thrombin in diluted serum can be detected at the concentration as low as 250 pM. In contrast, in a traditional molecular size-dependent FA assay, the thrombin spiked in diluted serum cannot induce reliable FA change even at a 256-fold higher concentration (64 nM). The results clearly show that the FA reduction approach has a dramatically enhanced specificity against target protein and high sensitivity in complex medium and is applicable to the no-separation based detection of proteins in biological matrixes.

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“…The biotinylated DNA aptamers were synthesized and purified by Sangon Biotech (Shanghai, China, http://www.sangon.com). The DNA aptamers for thrombin had the following sequences, 5′-A GTC CGT GGT AGG GCA GGT TGG GGT GAC T-3′ (Apt-29) and 5′-GGT TGG TGT GGT TGG-3′ (Apt-15) [17,18]. Biotin was labeled on 5′ end in all oligonucleotides.…”

Section: Experimental Reagents and Apparatusmentioning

confidence: 99%

“…Aptamers are selected for inhibition of thrombin activity in hydrolysis of fibrinogen to control the function of thrombin in blood clotting [17,18]. The specific binding of aptamers to thrombin also allows the use of aptamers as affinity ligands in assay developments for thrombin detection [7].…”

Section: Choice Of Aptamersmentioning

confidence: 99%

“…Two DNA aptamers are widely used in these aptamer-based assays for thrombin. A 15-nt aptamer (Apt-15, 5′-GGT TGG TGT GGT TGG-3′) [17] binds with the fibrinogen binding site of thrombin with a dissociation constant (K d ) around 100 nM, and another 29-nt aptamer (Apt-29, 5′-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3′) [18] can bind to the heparinbinding site of thrombin with a dissociation constant (K d ) around 0.5 nM. White et al reported an RNA aptamer (Toggle-25, 5′-GGG AAC AAA GCU GAA GUA CUU ACC C-3′) in 2001 [19].…”

Section: Introductionmentioning

confidence: 99%

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Microplate based assay for thrombin detection using an RNA aptamer as affinity ligand and cleavage of a chromogenic or a fluorogenic peptide substrate

Líu

Zhao

2016

Microchim Acta

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The authors report on a microplate based thrombin assay by using an RNA aptamer (Toggle-25) as the affinity ligand. The aptamer is nuclease-resistant because it contains nucleobases modified with 2′-deoxyfluoro saccharides. The aptamer is coated on the surface of the wells of microplates where it captures thrombin from a sample. Following a washing step, a chromogenic or a fluorogenic peptide substrate is added which is cleaved by the captured thrombin to form a yellow product (with absorbance at 405 nm) or a blue fluorescent product (with an emission maximum at 444 nm) after incubation at 37°C for 2 h. The assay enables thrombin to be determined with a 10 fM detection limit when using the fluorogenic substrate. The assay is selective in that several other proteins do not interfere. The assay can be applied to the determination of thrombin in diluted human serum and plasma.

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Oligonucleotide inhibitors of human thrombin that bind distinct epitopes

Cited by 791 publications

References 31 publications

Protein detection via direct enzymatic amplification of short DNA aptamers

Protein detection via direct enzymatic amplification of short DNA aptamers

Fluorescence Anisotropy Reduction of Allosteric Aptamer for Sensitive and Specific Protein Signaling

Microplate based assay for thrombin detection using an RNA aptamer as affinity ligand and cleavage of a chromogenic or a fluorogenic peptide substrate

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