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Selective light-triggered release of DNA from gold nanorods switches blood clotting on and off.

de Puig H, Cifuentes Rius A, Flemister D, Baxamusa SH, Hamad-Schifferli K - PLoS ONE (2013)

Bottom Line: We then release the complementary DNA as an antidote from the other NR, reversing the effect of the aptamer and restoring blood clotting.One challenge for nanobiotechnology is the bio-nano interface, where coronas of weakly adsorbed proteins can obscure biomolecular function.We exploit these adsorbed proteins to increase aptamer and antidote loading on the nanorods.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

ABSTRACT
Blood clotting is a precise cascade engineered to form a clot with temporal and spatial control. Current control of blood clotting is achieved predominantly by anticoagulants and thus inherently one-sided. Here we use a pair of nanorods (NRs) to provide a two-way switch for the blood clotting cascade by utilizing their ability to selectively release species on their surface under two different laser excitations. We selectively trigger release of a thrombin binding aptamer from one nanorod, inhibiting blood clotting and resulting in increased clotting time. We then release the complementary DNA as an antidote from the other NR, reversing the effect of the aptamer and restoring blood clotting. Thus, the nanorod pair acts as an on/off switch. One challenge for nanobiotechnology is the bio-nano interface, where coronas of weakly adsorbed proteins can obscure biomolecular function. We exploit these adsorbed proteins to increase aptamer and antidote loading on the nanorods.

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TBA and antidote affect coagulation in whole human blood.a) Schematic of coronas made from human serum (HS) loaded with NRs and TBA (NR-HS-TBA) + coronas loaded with NBs and antidote (NB-HS-antidote). 800 nm laser irradiation melts the NRs, triggering release of TBA from the coronas, which inhibits thrombin and causes blood coagulation times to increase. Following this, 1100 nm laser irradiation melts the NBs, triggering release of the DNA antidote from the corona. The antidote forms a double-stranded hybrid with TBA, thus restoring thrombin activity and blood coagulation. Fluorescently labeled TBA has a sequence of 5’ GGTTGGTGTGGTTGG-TMR 3’. The fluorescently labeled antidote has the complementary sequence 5’ CCAACCACACCAACC-FAM 3’. Clotting time (tplasma) for a thrombin test using 10 nM thrombin measured by a coagulometer with b) TBA, for c) 500 nM TBA + varying antidote from [anti]  =  0 to 1000 nM (anti/TBA  =  0 to 2.0).
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pone-0068511-g001: TBA and antidote affect coagulation in whole human blood.a) Schematic of coronas made from human serum (HS) loaded with NRs and TBA (NR-HS-TBA) + coronas loaded with NBs and antidote (NB-HS-antidote). 800 nm laser irradiation melts the NRs, triggering release of TBA from the coronas, which inhibits thrombin and causes blood coagulation times to increase. Following this, 1100 nm laser irradiation melts the NBs, triggering release of the DNA antidote from the corona. The antidote forms a double-stranded hybrid with TBA, thus restoring thrombin activity and blood coagulation. Fluorescently labeled TBA has a sequence of 5’ GGTTGGTGTGGTTGG-TMR 3’. The fluorescently labeled antidote has the complementary sequence 5’ CCAACCACACCAACC-FAM 3’. Clotting time (tplasma) for a thrombin test using 10 nM thrombin measured by a coagulometer with b) TBA, for c) 500 nM TBA + varying antidote from [anti]  =  0 to 1000 nM (anti/TBA  =  0 to 2.0).

Mentions: Thrombin inhibitors are of great interest as candidates for anticoagulants because thrombin, which cleaves fibrinogen into fibrin to form the clot, is at the apex of the clotting cascade [11], [12]. We used ssDNA thrombin binding aptamers (TBA) to inhibit thrombin and consequently coagulation. We then used complementary DNA as an antidote because it can reverse TBA’s effect by base-pairing with it (Figure 1a). Selective excitation of two different NRs to release TBA and its antidote enables the pair to act as an on/off switch for coagulation.


Selective light-triggered release of DNA from gold nanorods switches blood clotting on and off.

de Puig H, Cifuentes Rius A, Flemister D, Baxamusa SH, Hamad-Schifferli K - PLoS ONE (2013)

TBA and antidote affect coagulation in whole human blood.a) Schematic of coronas made from human serum (HS) loaded with NRs and TBA (NR-HS-TBA) + coronas loaded with NBs and antidote (NB-HS-antidote). 800 nm laser irradiation melts the NRs, triggering release of TBA from the coronas, which inhibits thrombin and causes blood coagulation times to increase. Following this, 1100 nm laser irradiation melts the NBs, triggering release of the DNA antidote from the corona. The antidote forms a double-stranded hybrid with TBA, thus restoring thrombin activity and blood coagulation. Fluorescently labeled TBA has a sequence of 5’ GGTTGGTGTGGTTGG-TMR 3’. The fluorescently labeled antidote has the complementary sequence 5’ CCAACCACACCAACC-FAM 3’. Clotting time (tplasma) for a thrombin test using 10 nM thrombin measured by a coagulometer with b) TBA, for c) 500 nM TBA + varying antidote from [anti]  =  0 to 1000 nM (anti/TBA  =  0 to 2.0).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3722233&req=5

pone-0068511-g001: TBA and antidote affect coagulation in whole human blood.a) Schematic of coronas made from human serum (HS) loaded with NRs and TBA (NR-HS-TBA) + coronas loaded with NBs and antidote (NB-HS-antidote). 800 nm laser irradiation melts the NRs, triggering release of TBA from the coronas, which inhibits thrombin and causes blood coagulation times to increase. Following this, 1100 nm laser irradiation melts the NBs, triggering release of the DNA antidote from the corona. The antidote forms a double-stranded hybrid with TBA, thus restoring thrombin activity and blood coagulation. Fluorescently labeled TBA has a sequence of 5’ GGTTGGTGTGGTTGG-TMR 3’. The fluorescently labeled antidote has the complementary sequence 5’ CCAACCACACCAACC-FAM 3’. Clotting time (tplasma) for a thrombin test using 10 nM thrombin measured by a coagulometer with b) TBA, for c) 500 nM TBA + varying antidote from [anti]  =  0 to 1000 nM (anti/TBA  =  0 to 2.0).
Mentions: Thrombin inhibitors are of great interest as candidates for anticoagulants because thrombin, which cleaves fibrinogen into fibrin to form the clot, is at the apex of the clotting cascade [11], [12]. We used ssDNA thrombin binding aptamers (TBA) to inhibit thrombin and consequently coagulation. We then used complementary DNA as an antidote because it can reverse TBA’s effect by base-pairing with it (Figure 1a). Selective excitation of two different NRs to release TBA and its antidote enables the pair to act as an on/off switch for coagulation.

Bottom Line: We then release the complementary DNA as an antidote from the other NR, reversing the effect of the aptamer and restoring blood clotting.One challenge for nanobiotechnology is the bio-nano interface, where coronas of weakly adsorbed proteins can obscure biomolecular function.We exploit these adsorbed proteins to increase aptamer and antidote loading on the nanorods.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

ABSTRACT
Blood clotting is a precise cascade engineered to form a clot with temporal and spatial control. Current control of blood clotting is achieved predominantly by anticoagulants and thus inherently one-sided. Here we use a pair of nanorods (NRs) to provide a two-way switch for the blood clotting cascade by utilizing their ability to selectively release species on their surface under two different laser excitations. We selectively trigger release of a thrombin binding aptamer from one nanorod, inhibiting blood clotting and resulting in increased clotting time. We then release the complementary DNA as an antidote from the other NR, reversing the effect of the aptamer and restoring blood clotting. Thus, the nanorod pair acts as an on/off switch. One challenge for nanobiotechnology is the bio-nano interface, where coronas of weakly adsorbed proteins can obscure biomolecular function. We exploit these adsorbed proteins to increase aptamer and antidote loading on the nanorods.

Show MeSH