Probing and quantifying DNA–protein interactions with asymmetrical flow field-flow fractionation

Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA-protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16nM and ∼57nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems

Flow Field Flow Fractionation Method Development for Applied Bioanalysis

Flow field flow fractionation (F4) and asymmetric F4 (AF4) are open channel separation instruments relying on an axial-flow and a perpendicular cross-flow to separate analytes based on hydrodynamic radius. The cross-flow provides enough force to simultaneously separate non-binding analytes based on size and remove non-specific binding giving the F4 potential as a method for bioanalysis. With the desire to apply F4 in bioanalysis, a need to understand how a complex sample, containing a variety of biomolecules and nanomaterials, would behave in the system was created. Changes in carrier solution (CS) and binding buffer (BB) composition such as ionic strength, cation type, and pH affect on particle and protein recoveries, and on the binding between protein and a ssDNA were investigated. Proteins required higher ionic strength to prevent adsorption in the channel, while nanoparticles had the opposite trend with increased ionic strength causing lower recoveries. No effect was seen from varying the BB, but the CS had played a significant role. During AF4's unique focusing step, the BB is replaced with the CS rapidly, indicating that on-line incubation is a possibility for F4 bioanalysis reducing the amount of sample handling time. The presence of MgCl2 in the CS, which plays an important role in DNA folding, was necessary for binding to occur. An experimental detection limit of 33 nM was achieved for immunoglobulin E, limited by the labeling of one fluorophore per protein.The study on protein-ssDNA analysis was expanded to calculate the dissociation constant (Kd) of the model system which was in very good agreement with Kd values obtained by other methods. A variety of ssDNA was investigated to determine the affect different lengths and secondary structures had on the recovery and retention times in F4. With the ability to possibly bind both intramoleculary and intermoleculary, ssDNA showed unique elution profiles from more globular analytes. Folding equilibriums were calculated for ssDNA with known secondary structure by comparing the elution times of linear DNA that was unlikely to form intramolecular bonds. This work has shown the versatility F4/AF4 could have for future applications of ssDNA as labeling agents

Dissociation-based screening of nanoparticle-protein interaction via flow field-flow fractionation.

A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles' subsequent behaviors inside the biological systems. For proteins bound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. In the present study, a method to differentiate the corona proteins based on their relative dissociation rates from the NPs was developed, employing flow field-flow fraction (F4) in combination with centrifugation. The proteins bound to the superparamagnetic iron oxide NPs (SPION) present in an IgG/albumin depleted serum were isolated via collection of the SPIONs by either F4 or centrifugation. They were subsequently analyzed by LC-MS/MS and identified. Because the SPION-protein complexes injected to F4 dissociated continuously under the nonequilibrium separation condition, only the proteins with slow enough dissociation rates would be collected with the NPs in the eluent of F4. However, in centrifugation, proteins with good affinity to the SPIONs were collected regardless of the dissociation rates of the complexes. In both cases, the nonbinding ones were washed off. Capillary electrophoresis and circular dichroism were employed to verify the binding situations of a few SPION-protein interactions, confirming the effectiveness of our method. Our results support that our method can screen for proteins binding to NPs with fast on-and-off rates, which should be the ones quickly exchanging with the free matrix proteins when the NPs are exposed to a new biological media. Thus, our method will be useful for investigation of the temporal profile of protein corona and its evolution in biological matrices as well as for high-throughput analysis of the dynamic feature of protein corona related to particle properties

Dissociation-based screening of nanoparticle-protein interaction via flow field-flow fractionation.

A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles' subsequent behaviors inside the biological systems. For proteins bound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. In the present study, a method to differentiate the corona proteins based on their relative dissociation rates from the NPs was developed, employing flow field-flow fraction (F4) in combination with centrifugation. The proteins bound to the superparamagnetic iron oxide NPs (SPION) present in an IgG/albumin depleted serum were isolated via collection of the SPIONs by either F4 or centrifugation. They were subsequently analyzed by LC-MS/MS and identified. Because the SPION-protein complexes injected to F4 dissociated continuously under the nonequilibrium separation condition, only the proteins with slow enough dissociation rates would be collected with the NPs in the eluent of F4. However, in centrifugation, proteins with good affinity to the SPIONs were collected regardless of the dissociation rates of the complexes. In both cases, the nonbinding ones were washed off. Capillary electrophoresis and circular dichroism were employed to verify the binding situations of a few SPION-protein interactions, confirming the effectiveness of our method. Our results support that our method can screen for proteins binding to NPs with fast on-and-off rates, which should be the ones quickly exchanging with the free matrix proteins when the NPs are exposed to a new biological media. Thus, our method will be useful for investigation of the temporal profile of protein corona and its evolution in biological matrices as well as for high-throughput analysis of the dynamic feature of protein corona related to particle properties

Probing and quantifying DNA-protein interactions with asymmetrical flow field-flow fractionation.

Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA-protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16nM and ∼57nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems

Dissociation-Based Screening of Nanoparticle–Protein Interaction via Flow Field-Flow Fractionation

A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles’ subsequent behaviors inside the biological systems. For proteins bound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. In the present study, a method to differentiate the corona proteins based on their relative dissociation rates from the NPs was developed, employing flow field-flow fraction (F4) in combination with centrifugation. The proteins bound to the superparamagnetic iron oxide NPs (SPION) present in an IgG/albumin depleted serum were isolated via collection of the SPIONs by either F4 or centrifugation. They were subsequently analyzed by LC-MS/MS and identified. Because the SPION-protein complexes injected to F4 dissociated continuously under the nonequilibrium separation condition, only the proteins with slow enough dissociation rates would be collected with the NPs in the eluent of F4. However, in centrifugation, proteins with good affinity to the SPIONs were collected regardless of the dissociation rates of the complexes. In both cases, the nonbinding ones were washed off. Capillary electrophoresis and circular dichroism were employed to verify the binding situations of a few SPION-protein interactions, confirming the effectiveness of our method. Our results support that our method can screen for proteins binding to NPs with fast on-and-off rates, which should be the ones quickly exchanging with the free matrix proteins when the NPs are exposed to a new biological media. Thus, our method will be useful for investigation of the temporal profile of protein corona and its evolution in biological matrices as well as for high-throughput analysis of the dynamic feature of protein corona related to particle properties

Dissociation-Based Screening of Nanoparticle–Protein Interaction via Flow Field-Flow Fractionation

A protein corona will be formed on nanoparticles (NPs) entering a biological matrix, which can influence particles’ subsequent behaviors inside the biological systems. For proteins bound stably to the NPs, they can exhibit different association/dissociation rates. The binding kinetics could affect interaction of the NPs with cell surface receptors and possibly contribute to the outcomes of NPs uptake. In the present study, a method to differentiate the corona proteins based on their relative dissociation rates from the NPs was developed, employing flow field-flow fraction (F4) in combination with centrifugation. The proteins bound to the superparamagnetic iron oxide NPs (SPION) present in an IgG/albumin depleted serum were isolated via collection of the SPIONs by either F4 or centrifugation. They were subsequently analyzed by LC-MS/MS and identified. Because the SPION-protein complexes injected to F4 dissociated continuously under the non-equilibrium separation condition, only the proteins with slow enough dissociation rates would be collected with the NPs in the eluent of F4. However, in centrifugation, proteins with good affinity to the SPIONs were collected regardless of the dissociation rates of the complexes. In both cases, the non-binding ones were washed off. Capillary electrophoresis and circular dichroism were employed to verify the binding situations of a few SPION-protein interactions, confirming the effectiveness of our method. Our results support that our method can screen for proteins binding to NPs with fast on-and-off rates, which should be the ones quickly exchanging with the free matrix proteins when the NPs are exposed to a new biological media. Thus, our method will be useful for investigation of the temporal profile of protein corona and its evolution in biological matrices, as well as for high-throughput analysis of the dynamic feature of protein corona related to particle properties