Limits...
Kinetic and thermodynamic characterization of single-mismatch discrimination using single-molecule imaging.

Gunnarsson A, Jönsson P, Zhdanov VP, Höök F - Nucleic Acids Res. (2009)

Bottom Line: The liposomes, acting as signal enhancer elements, enabled the number of binding events as well as the residence time for high affinity binders (K(d) < 1 nM, k(off) < 0.01 s(-1)) to be collected under equilibrium conditions at low pM concentrations.The mismatch discrimination obtained from the residence time data was shown to be concentration and temperature independent in intervals of 1-100 pM and 23-46 degrees C, respectively.This suggests the method as a robust means for detection of point mutations at low target concentrations in, for example, single nucleotide polymorphism (SNP) analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Physics, Chalmers University of Technology, SE-41296 Gothenburg, Sweden.

ABSTRACT
A single-molecule detection setup based on total internal reflection fluorescence (TIRF) microscopy has been used to investigate association and dissociation kinetics of unlabeled 30mer DNA strands. Single-molecule sensitivity was accomplished by letting unlabeled DNA target strands mediate the binding of DNA-modified and fluorescently labeled liposomes to a DNA-modified surface. The liposomes, acting as signal enhancer elements, enabled the number of binding events as well as the residence time for high affinity binders (K(d) < 1 nM, k(off) < 0.01 s(-1)) to be collected under equilibrium conditions at low pM concentrations. The mismatch discrimination obtained from the residence time data was shown to be concentration and temperature independent in intervals of 1-100 pM and 23-46 degrees C, respectively. This suggests the method as a robust means for detection of point mutations at low target concentrations in, for example, single nucleotide polymorphism (SNP) analysis.

Show MeSH

Related in: MedlinePlus

(A) Schematic illustration of the single-molecule sensing template. An unlabeled DNA target mediates the binding of rhodamine-labeled liposomes (Ø ∼100 nm) modified with, on average, one DNA duplex with a single stranded sticky-end (15 bases) to a TIR-illuminated surface modified with single stranded DNA (15 bases). The residence time of the liposomes on the surface is monitored for the kinetic analysis. The silicon dioxide surface is modified by self-assembly of biotinylated copolymer (PLL-g-PEG/PLL-g-PEGbiotin) followed by binding of Neutravidin conjugated to biotinylated single stranded DNA. (B) Time trace of binding and dissociation of a single liposome on the surface. The purple and yellow arrows mark the binding and dissociation event, respectively. (C) Microscopy snapshot of a subsection of the illuminated area during analysis. Green crosses indicate bound liposomes. Yellow rings show liposomes detaching from the surface in the subsequent frame. Purple crosses indicate new liposomes binding in the current frame. Field of view of the subsection is 40 × 40 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2724293&req=5

Figure 1: (A) Schematic illustration of the single-molecule sensing template. An unlabeled DNA target mediates the binding of rhodamine-labeled liposomes (Ø ∼100 nm) modified with, on average, one DNA duplex with a single stranded sticky-end (15 bases) to a TIR-illuminated surface modified with single stranded DNA (15 bases). The residence time of the liposomes on the surface is monitored for the kinetic analysis. The silicon dioxide surface is modified by self-assembly of biotinylated copolymer (PLL-g-PEG/PLL-g-PEGbiotin) followed by binding of Neutravidin conjugated to biotinylated single stranded DNA. (B) Time trace of binding and dissociation of a single liposome on the surface. The purple and yellow arrows mark the binding and dissociation event, respectively. (C) Microscopy snapshot of a subsection of the illuminated area during analysis. Green crosses indicate bound liposomes. Yellow rings show liposomes detaching from the surface in the subsequent frame. Purple crosses indicate new liposomes binding in the current frame. Field of view of the subsection is 40 × 40 µm.

Mentions: Imaging techniques provide a solution to the problem connected with sequential readout, but in order to reach single-molecule sensitivity, the target molecules must generally be fluorescently labeled. Due to rapid photobleaching, this restricts the detection time window to a few seconds (26,27) and can thus only be applied to low affinity binders (koff > 0.1 s−1). Using a sandwich format similar to that utilized by Cao et al. for detection of DNA and RNA targets (28), we recently demonstrated that TIRF microscopy can provide parallel detection of high affinity (koff < 0.01 s−1) single hybridization events with a LOD in the low fM regime. This was accomplished by letting unlabeled DNA targets mediate the binding of fluorescently labeled liposomes to a DNA-modified surface (29). In the present work, we extend the concept (schematically illustrated in Figure 1) by performing an analysis in equilibrium of single binding events. In this way, determination of the equilibrium dissociation constant, Kd, and a thermodynamic analysis of the dissociation rate constant, koff, are shown possible from a single injection of DNA targets at low pM concentrations. Operation under equilibrium binding conditions reduces problems related to mass-transport limitations, and the single-molecule sensitivity makes the concept compatible with low surface-probe densities (<0.1 pmol/cm2). Particular focus is put on the capacity of the assay to discriminate fully complementary from single mismatch sequences. The potential of single-mismatch discrimination was evaluated in a range of target concentrations (1–100 pM) and in a broad temperature interval (23–46°C). We also propose an alternative kinetic and thermodynamic analysis of this type of data, which relies on the ratio between the number of detaching liposomes and the integrated number of bound liposomes at equilibrium binding conditions.Figure 1.


Kinetic and thermodynamic characterization of single-mismatch discrimination using single-molecule imaging.

Gunnarsson A, Jönsson P, Zhdanov VP, Höök F - Nucleic Acids Res. (2009)

(A) Schematic illustration of the single-molecule sensing template. An unlabeled DNA target mediates the binding of rhodamine-labeled liposomes (Ø ∼100 nm) modified with, on average, one DNA duplex with a single stranded sticky-end (15 bases) to a TIR-illuminated surface modified with single stranded DNA (15 bases). The residence time of the liposomes on the surface is monitored for the kinetic analysis. The silicon dioxide surface is modified by self-assembly of biotinylated copolymer (PLL-g-PEG/PLL-g-PEGbiotin) followed by binding of Neutravidin conjugated to biotinylated single stranded DNA. (B) Time trace of binding and dissociation of a single liposome on the surface. The purple and yellow arrows mark the binding and dissociation event, respectively. (C) Microscopy snapshot of a subsection of the illuminated area during analysis. Green crosses indicate bound liposomes. Yellow rings show liposomes detaching from the surface in the subsequent frame. Purple crosses indicate new liposomes binding in the current frame. Field of view of the subsection is 40 × 40 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724293&req=5

Figure 1: (A) Schematic illustration of the single-molecule sensing template. An unlabeled DNA target mediates the binding of rhodamine-labeled liposomes (Ø ∼100 nm) modified with, on average, one DNA duplex with a single stranded sticky-end (15 bases) to a TIR-illuminated surface modified with single stranded DNA (15 bases). The residence time of the liposomes on the surface is monitored for the kinetic analysis. The silicon dioxide surface is modified by self-assembly of biotinylated copolymer (PLL-g-PEG/PLL-g-PEGbiotin) followed by binding of Neutravidin conjugated to biotinylated single stranded DNA. (B) Time trace of binding and dissociation of a single liposome on the surface. The purple and yellow arrows mark the binding and dissociation event, respectively. (C) Microscopy snapshot of a subsection of the illuminated area during analysis. Green crosses indicate bound liposomes. Yellow rings show liposomes detaching from the surface in the subsequent frame. Purple crosses indicate new liposomes binding in the current frame. Field of view of the subsection is 40 × 40 µm.
Mentions: Imaging techniques provide a solution to the problem connected with sequential readout, but in order to reach single-molecule sensitivity, the target molecules must generally be fluorescently labeled. Due to rapid photobleaching, this restricts the detection time window to a few seconds (26,27) and can thus only be applied to low affinity binders (koff > 0.1 s−1). Using a sandwich format similar to that utilized by Cao et al. for detection of DNA and RNA targets (28), we recently demonstrated that TIRF microscopy can provide parallel detection of high affinity (koff < 0.01 s−1) single hybridization events with a LOD in the low fM regime. This was accomplished by letting unlabeled DNA targets mediate the binding of fluorescently labeled liposomes to a DNA-modified surface (29). In the present work, we extend the concept (schematically illustrated in Figure 1) by performing an analysis in equilibrium of single binding events. In this way, determination of the equilibrium dissociation constant, Kd, and a thermodynamic analysis of the dissociation rate constant, koff, are shown possible from a single injection of DNA targets at low pM concentrations. Operation under equilibrium binding conditions reduces problems related to mass-transport limitations, and the single-molecule sensitivity makes the concept compatible with low surface-probe densities (<0.1 pmol/cm2). Particular focus is put on the capacity of the assay to discriminate fully complementary from single mismatch sequences. The potential of single-mismatch discrimination was evaluated in a range of target concentrations (1–100 pM) and in a broad temperature interval (23–46°C). We also propose an alternative kinetic and thermodynamic analysis of this type of data, which relies on the ratio between the number of detaching liposomes and the integrated number of bound liposomes at equilibrium binding conditions.Figure 1.

Bottom Line: The liposomes, acting as signal enhancer elements, enabled the number of binding events as well as the residence time for high affinity binders (K(d) < 1 nM, k(off) < 0.01 s(-1)) to be collected under equilibrium conditions at low pM concentrations.The mismatch discrimination obtained from the residence time data was shown to be concentration and temperature independent in intervals of 1-100 pM and 23-46 degrees C, respectively.This suggests the method as a robust means for detection of point mutations at low target concentrations in, for example, single nucleotide polymorphism (SNP) analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Physics, Chalmers University of Technology, SE-41296 Gothenburg, Sweden.

ABSTRACT
A single-molecule detection setup based on total internal reflection fluorescence (TIRF) microscopy has been used to investigate association and dissociation kinetics of unlabeled 30mer DNA strands. Single-molecule sensitivity was accomplished by letting unlabeled DNA target strands mediate the binding of DNA-modified and fluorescently labeled liposomes to a DNA-modified surface. The liposomes, acting as signal enhancer elements, enabled the number of binding events as well as the residence time for high affinity binders (K(d) < 1 nM, k(off) < 0.01 s(-1)) to be collected under equilibrium conditions at low pM concentrations. The mismatch discrimination obtained from the residence time data was shown to be concentration and temperature independent in intervals of 1-100 pM and 23-46 degrees C, respectively. This suggests the method as a robust means for detection of point mutations at low target concentrations in, for example, single nucleotide polymorphism (SNP) analysis.

Show MeSH
Related in: MedlinePlus