An Ambiguous Structure of a DNA 15-mer Thrombin Complex

Padmanabhan, K.; Tulinsky, A.

doi:10.1107/s0907444995013977

Cited by 167 publications

(210 citation statements)

References 29 publications

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“…Conceptual visualization of the aptamer based sensing platform with fluorescently labeled aptamers (TBA1 with green and TBA2 with red label) forming a sandwich with thrombin on patterned glass surfaces, i.e., spotted microarray (left) and microcontact printed array (right). Molecular structures for this scheme were taken from the RCSB protein data bank (PDB ID: 1HAO) 39 and visualized in VMD 1.9.1. the addition of succinic anhydride dissolved in DMSO (final concentration: 10 mg/mL) the amino surface was carboxylated in a ring-opening reaction. After 30 min, the samples were rinsed with DI water in an ultrasonic bath for 3 min and dried with N 2 .…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay

Römhildt

Pahlke²,

Zörgiebel³

et al. 2013

ACS Appl. Mater. Interfaces

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Here we propose a platform for the detection of unlabeled human α-thrombin down to the picomolar range in a fluorescence-based aptamer assay. In this concept, thrombin is captured between two different thrombin binding aptamers, TBA1 (15mer) and TBA2 (29mer), each labeled with a specific fluorescent dye. One aptamer is attached to the surface, the second one is in solution and recognizes surface-captured thrombin. To improve the limit of detection and the comparability of measurements, we employed and compared two approaches to pattern the chip substrate−microcontact printing of organosilanes onto bare glass slides, and controlled printing of the capture aptamer TBA1 in arrays onto functionalized glass substrates using a nanoplotter device. The parallel presence of functionalized and control areas acts as an internal reference. We demonstrate that both techniques enable the detection of thrombin concentrations in a wide range from 0.02 to 200 nM with a detection limit at 20 pM. Finally, the developed method could be transferred to any substrate to probe different targets that have two distinct possible receptors without the need for direct target labeling.

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Section: ■ Experimental Sectionmentioning

confidence: 99%

Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay

Römhildt

Pahlke²,

Zörgiebel³

et al. 2013

ACS Appl. Mater. Interfaces

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“…The crystal structure of a DNA aptamer bound to thrombin was published in 1993 and 1996, and the structure provided the first example of how a DNA aptamer could interact with a protein that does not naturally recognize a nucleic acid (Bock et al 1992;Padmanabhan et al 1993;Padmanabhan and Tulinsky 1996) Moreover, mutagenesis studies on thrombin confirmed that the DNA aptamer binds exosite 1 (Padmanabhan et al 1993). Similar thrombin mutagenesis studies indicated that the RNA aptamer TOG25 binds to thrombin near exosite 2 (Jeter et al 2004).…”

Section: Dna and Rna Aptamers Bind To Thrombin On Different Exositesmentioning

confidence: 99%

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin

Nimjee

Oney²,

Volovyk³

et al. 2009

RNA

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Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin's active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach.

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“…In structures of aptamers bound to nucleic acid binding proteins, the aptamers bind to the nucleic acid binding site and often mimic naturally occurring interactions (Convery et al 1998;Jaeger et al 1998;Cox et al 2002;Huang et al 2003;Ghosh et al 2004). The crystal structure of a DNA aptamer bound to thrombin has been determined (Padmanabhan et al 1993;Padmanabhan and Tulinsky 1996). This is the only crystal structure so far determined of an aptamer in complex with a protein that does not naturally bind nucleic acid.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of an RNA aptamer bound to thrombin

Long¹,

Long²,

White³

et al. 2008

RNA

145

151

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Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 Å resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.

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An Ambiguous Structure of a DNA 15-mer Thrombin Complex

Cited by 167 publications

References 29 publications

Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay

Patterned Biochemical Functionalization Improves Aptamer-Based Detection of Unlabeled Thrombin in a Sandwich Assay

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin

Crystal structure of an RNA aptamer bound to thrombin

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