Novel strategy for DNA aptamers inhibiting enzymatic activity using algorithm mimicking evolution

Okumura, Yuji; Sumikura, Koichi; Karube, Isao

doi:10.1093/nass/3.1.205

Cited by 8 publications

(11 citation statements)

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“…For the thrombin detection system, we used a 31-mer thrombin aptamer [24], which contained a 5-bp stem and a 15-mer loop sequence as the thrombin recognition element [19] and a 19-, 15-or 11-mer complementary strand of the stem-loop sequence, to trap the thrombin aptamer. For the IgE detection system, we used a 31-mer IgE aptamer [25], together with a 14-mer complementary strand that hybridizes to the 5'-end of the IgE aptamer.…”

Section: Oligonucleotidesmentioning

confidence: 99%

An Aptamer‐Based Bound/Free Separation System for Protein Detection

Fukasawa

Yoshida

Yamazaki³

et al. 2009

Electroanalysis

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Aptamer hybridizes with its complementary strand. However, the complementary strand has difficulties to hybridize with the aptamer bound to a target because the aptamer forms higher-order structures. Exploiting this property, we developed simple bound/free separation systems for thrombin and IgE detection. The complementary strand was immobilized onto beads and the aptamer was labeled with pyrroquinoline quinone glucose dehydrogenase (PQQGDH). In the absence of a target, the aptamer is trapped by beads, whereas in the presence of a target, the aptamer bound to the target is not trapped. Thus the aptamer-target complexes can be recovered easily and detected by PQQGDH activity. This system allow the detection of 270 pM thrombin and 1 nM IgE.

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Section: Oligonucleotidesmentioning

confidence: 99%

An Aptamer‐Based Bound/Free Separation System for Protein Detection

Fukasawa

Yoshida

Yamazaki³

et al. 2009

Electroanalysis

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“…Some of the outcome of this study were presented at the Third International Symposium on Nucleic Acids Chemistry held at Sapporo, Japan in 2003 and it had already been published as an abstract (24). …”

Section: Introductionmentioning

confidence: 99%

A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm

Ikebukuro

Okumura²,

Sumikura³

et al. 2005

Nucleic Acids Research

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Thrombin-inhibiting DNA aptamers have already been obtained through the systematic evolution of ligands by exponential enrichment (SELEX). However, SELEX is a method that screens DNA aptamers that bind to their target molecules, and it sometimes fails to screen good inhibitors. Therefore, it is necessary to develop a method of screening DNA aptamers based on their inhibitory effects on the target molecules. We developed a novel method of detecting aptamers using an evolution-mimicking algorithm, and we applied it to the search of new aptamers which inhibit thrombin. First, we randomly designed and synthesized ten 15mer oligonucleotides presumed to form G-quartet structures, and then measured their thrombin-inhibiting activities. The aptamers showing high inhibitory activity were selected, and we shuffled and mutated those sequences in silico to generate 10 new sequences of next-generation aptamers. After repeating the cycle five times, we successfully obtained the same aptamers reported previously, and they showed high inhibitory activity. In addition, we added 8mer oligonucleotides to both the 5′ and the 3′ end of the selected 15mer aptamers, and then repeated the evolution in silico. After two cycles, we were able to obtain aptamers with higher inhibitory activity than that of the 15mer aptamers.

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“…In particular, a 31-base aptamer (31TBA) was identified that is structurally similar to 15TBA but contains additional flanking sequences (Fig. 1) [27,48]. It was reported that at a very high concentration (30 mmol/l), this aptamer increased the in-vitro thrombin time in plasma by 1.75-fold compared with 15TBA [27].…”

Section: Discussionmentioning

confidence: 99%

Thrombin inhibitors based on single-stranded DNA aptamers

Gribkova

Спиридонова²,

Gorbatenko³

et al. 2014

Blood Coagulation &Amp; Fibrinolysis

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Investigation of inhibitory effect of two single-stranded DNA thrombin-inhibiting aptamers (15TBA and 31TBA) on fibrin polymerization in fibrinogen solutions and comparison of anticoagulant properties of these aptamers by a new global coagulation test of thrombodynamics. Measurement of aptamers' functional stability in human plasma and blood in vitro in order to investigate the involvement of 3'-exonuclease in fast decrease of aptamers' functional activity in vivo. Thrombin inhibition activity was measured in a buffer system in vitro as effects of aptamers on fibrin polymerization. Anticoagulant activity was investigated by measuring the spatial clot growth rate in the presence of aptamers. The stability of aptamers during incubation in human plasma was investigated in vitro by measuring activated partial thromboplastin time. Both aptamers dose-dependently inhibit fibrin polymerization in a buffer solution (IC50=10 nm for 15TBA and 3 nm for 31TBA) and are effective anticoagulants in human plasma (IC50 for spatial clot growth rate decreasing are 9.5 μmol/l and 4.0 μmol/l for 15TBA and 31TBA, correspondingly). Both aptamers remain stable in plasma or whole blood in vitro for at least 4 h. It was shown that 31TBA was 2-3 times more effective than 15TBA. Both aptamers were stable in human plasma and whole blood in vitro. So, the 3'-exonuclease could not be the reason for fast decrease of aptamers' functional activity in vivo. The main role in the removal of oligonucleotides from the circulation is played obviously by the liver.

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Novel strategy for DNA aptamers inhibiting enzymatic activity using algorithm mimicking evolution

Cited by 8 publications

References 0 publications

An Aptamer‐Based Bound/Free Separation System for Protein Detection

An Aptamer‐Based Bound/Free Separation System for Protein Detection

A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm

Thrombin inhibitors based on single-stranded DNA aptamers

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