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Proximity extension of circular DNA aptamers with real-time protein detection.

Di Giusto DA, Wlassoff WA, Gooding JJ, Messerle BA, King GC - Nucleic Acids Res. (2005)

Bottom Line: The circular DNA architecture facilitates the integration of multiple functional elements into a single molecule: aptameric target recognition, nucleic acid hybridization specificity and rolling circle amplification.Real-time signal amplification and detection under isothermal conditions points towards potential clinical applications, with both fluorescent and bioelectronic methods of detection achieved.This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.

ABSTRACT
Multivalent circular aptamers or 'captamers' have recently been introduced through the merger of aptameric recognition functions with the basic principles of DNA nanotechnology. Aptamers have strong utility as protein-binding motifs for diagnostic applications, where their ease of discovery, thermal stability and low cost make them ideal components for incorporation into targeted protein assays. Here we report upon a property specific to circular DNA aptamers: their intrinsic compatibility with a highly sensitive protein detection method termed the 'proximity extension' assay. The circular DNA architecture facilitates the integration of multiple functional elements into a single molecule: aptameric target recognition, nucleic acid hybridization specificity and rolling circle amplification. Successful exploitation of these properties is demonstrated for the molecular analysis of thrombin, with the assay delivering a detection limit nearly three orders of magnitude below the dissociation constants of the two contributing aptamer-thrombin interactions. Real-time signal amplification and detection under isothermal conditions points towards potential clinical applications, with both fluorescent and bioelectronic methods of detection achieved. This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier.

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Related in: MedlinePlus

Aptamers for the detection of human thrombin by proximity extension. (A) Formation of the circular dumbbell CT-6 aptamer bearing a thrombin exosite II motif (underlined) plus an unstructured template loop (bold). (B) Linear LT-6 aptamer bearing a thrombin exosite I motif (underlined) plus a long tail with a complementary 3′-terminus (bold). Poly(dT) stretches of 22 and 48 nt in length are shown in parentheses. (C) Simultaneous binding of aptamers to thrombin primes DNA polymerase-mediated RCA. (D) Polyacrylamide gel (12%) with markers (M) followed by proximity-extension-mediated RCA products generated in the presence of (1) 0 pM thrombin, (2) 4000 pM thrombin and (3) 4000 pM thrombin with subsequent Taqα 1 restriction enzyme digestion. No product is generated in the absence of thrombin, while high molecular weight products are observed when thrombin is present. These products can be digested using Taqα 1 restriction enzyme into fragments displaying the expected sizes (36 and 31 nt for the 67 nt LT-6 template).
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fig1: Aptamers for the detection of human thrombin by proximity extension. (A) Formation of the circular dumbbell CT-6 aptamer bearing a thrombin exosite II motif (underlined) plus an unstructured template loop (bold). (B) Linear LT-6 aptamer bearing a thrombin exosite I motif (underlined) plus a long tail with a complementary 3′-terminus (bold). Poly(dT) stretches of 22 and 48 nt in length are shown in parentheses. (C) Simultaneous binding of aptamers to thrombin primes DNA polymerase-mediated RCA. (D) Polyacrylamide gel (12%) with markers (M) followed by proximity-extension-mediated RCA products generated in the presence of (1) 0 pM thrombin, (2) 4000 pM thrombin and (3) 4000 pM thrombin with subsequent Taqα 1 restriction enzyme digestion. No product is generated in the absence of thrombin, while high molecular weight products are observed when thrombin is present. These products can be digested using Taqα 1 restriction enzyme into fragments displaying the expected sizes (36 and 31 nt for the 67 nt LT-6 template).

Mentions: To construct the DNA components required to couple aptamer recognition with RCA, an aptamer against the heparin exosite II of human thrombin (31) and an unstructured template loop were first combined into a circular dumbbell architecture (24) (Figure 1A). To provide an aptamer capable of performing as a primer against this circular template, a second linear aptamer against thrombin exosite I (28) was engineered with a 3′ tail (Figure 1B). The tail of this linear aptamer was sufficiently long to bridge the distance to the circular aptamer only when both are bound to a target molecule. Upon target binding, the tail of the linear aptamer is then able to hybridize to the complementary unstructured loop of the circular aptamer, thus forming a primer for polymerase-mediated extension and isothermal RCA, converting the circular aptamer from a functional binding element into a structural template (Figure 1C). This RCA reaction should generate a product strand composed of the linear aptamer sequence at the 5′ end, followed by many concatenated sequences complementary to the circular aptamer template. The anticipated high molecular weight of this product was confirmed by the extremely low mobility of purified reaction samples following denaturing gel electrophoresis (Figure 1D, lane 2). Because the circular aptamer construct that acts as the template for RCA contains complementary sequences that form a central duplex, it is expected that the concatenated reaction product will contain corresponding regions of complementarity, generating multiple equivalent duplex structures in the nascent product. As these duplex regions can be designed to contain an RE recognition site, the identity of the RCA product and hence the mechanism of activity can be confirmed by the presence of correctly sized fragments upon restriction digestion of assay products (Figure 1D, lane 3).


Proximity extension of circular DNA aptamers with real-time protein detection.

Di Giusto DA, Wlassoff WA, Gooding JJ, Messerle BA, King GC - Nucleic Acids Res. (2005)

Aptamers for the detection of human thrombin by proximity extension. (A) Formation of the circular dumbbell CT-6 aptamer bearing a thrombin exosite II motif (underlined) plus an unstructured template loop (bold). (B) Linear LT-6 aptamer bearing a thrombin exosite I motif (underlined) plus a long tail with a complementary 3′-terminus (bold). Poly(dT) stretches of 22 and 48 nt in length are shown in parentheses. (C) Simultaneous binding of aptamers to thrombin primes DNA polymerase-mediated RCA. (D) Polyacrylamide gel (12%) with markers (M) followed by proximity-extension-mediated RCA products generated in the presence of (1) 0 pM thrombin, (2) 4000 pM thrombin and (3) 4000 pM thrombin with subsequent Taqα 1 restriction enzyme digestion. No product is generated in the absence of thrombin, while high molecular weight products are observed when thrombin is present. These products can be digested using Taqα 1 restriction enzyme into fragments displaying the expected sizes (36 and 31 nt for the 67 nt LT-6 template).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1074748&req=5

fig1: Aptamers for the detection of human thrombin by proximity extension. (A) Formation of the circular dumbbell CT-6 aptamer bearing a thrombin exosite II motif (underlined) plus an unstructured template loop (bold). (B) Linear LT-6 aptamer bearing a thrombin exosite I motif (underlined) plus a long tail with a complementary 3′-terminus (bold). Poly(dT) stretches of 22 and 48 nt in length are shown in parentheses. (C) Simultaneous binding of aptamers to thrombin primes DNA polymerase-mediated RCA. (D) Polyacrylamide gel (12%) with markers (M) followed by proximity-extension-mediated RCA products generated in the presence of (1) 0 pM thrombin, (2) 4000 pM thrombin and (3) 4000 pM thrombin with subsequent Taqα 1 restriction enzyme digestion. No product is generated in the absence of thrombin, while high molecular weight products are observed when thrombin is present. These products can be digested using Taqα 1 restriction enzyme into fragments displaying the expected sizes (36 and 31 nt for the 67 nt LT-6 template).
Mentions: To construct the DNA components required to couple aptamer recognition with RCA, an aptamer against the heparin exosite II of human thrombin (31) and an unstructured template loop were first combined into a circular dumbbell architecture (24) (Figure 1A). To provide an aptamer capable of performing as a primer against this circular template, a second linear aptamer against thrombin exosite I (28) was engineered with a 3′ tail (Figure 1B). The tail of this linear aptamer was sufficiently long to bridge the distance to the circular aptamer only when both are bound to a target molecule. Upon target binding, the tail of the linear aptamer is then able to hybridize to the complementary unstructured loop of the circular aptamer, thus forming a primer for polymerase-mediated extension and isothermal RCA, converting the circular aptamer from a functional binding element into a structural template (Figure 1C). This RCA reaction should generate a product strand composed of the linear aptamer sequence at the 5′ end, followed by many concatenated sequences complementary to the circular aptamer template. The anticipated high molecular weight of this product was confirmed by the extremely low mobility of purified reaction samples following denaturing gel electrophoresis (Figure 1D, lane 2). Because the circular aptamer construct that acts as the template for RCA contains complementary sequences that form a central duplex, it is expected that the concatenated reaction product will contain corresponding regions of complementarity, generating multiple equivalent duplex structures in the nascent product. As these duplex regions can be designed to contain an RE recognition site, the identity of the RCA product and hence the mechanism of activity can be confirmed by the presence of correctly sized fragments upon restriction digestion of assay products (Figure 1D, lane 3).

Bottom Line: The circular DNA architecture facilitates the integration of multiple functional elements into a single molecule: aptameric target recognition, nucleic acid hybridization specificity and rolling circle amplification.Real-time signal amplification and detection under isothermal conditions points towards potential clinical applications, with both fluorescent and bioelectronic methods of detection achieved.This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier.

View Article: PubMed Central - PubMed

Affiliation: School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.

ABSTRACT
Multivalent circular aptamers or 'captamers' have recently been introduced through the merger of aptameric recognition functions with the basic principles of DNA nanotechnology. Aptamers have strong utility as protein-binding motifs for diagnostic applications, where their ease of discovery, thermal stability and low cost make them ideal components for incorporation into targeted protein assays. Here we report upon a property specific to circular DNA aptamers: their intrinsic compatibility with a highly sensitive protein detection method termed the 'proximity extension' assay. The circular DNA architecture facilitates the integration of multiple functional elements into a single molecule: aptameric target recognition, nucleic acid hybridization specificity and rolling circle amplification. Successful exploitation of these properties is demonstrated for the molecular analysis of thrombin, with the assay delivering a detection limit nearly three orders of magnitude below the dissociation constants of the two contributing aptamer-thrombin interactions. Real-time signal amplification and detection under isothermal conditions points towards potential clinical applications, with both fluorescent and bioelectronic methods of detection achieved. This application elaborates the pleiotropic properties of circular DNA aptamers beyond the stability, potency and multitargeting characteristics described earlier.

Show MeSH
Related in: MedlinePlus