Limits...
Multiplexed microcolumn-based process for efficient selection of RNA aptamers.

Latulippe DR, Szeto K, Ozer A, Duarte FM, Kelly CV, Pagano JM, White BS, Shalloway D, Lis JT, Craighead HG - Anal. Chem. (2013)

Bottom Line: We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations.We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2.High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds.

View Article: PubMed Central - PubMed

Affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, United States.

ABSTRACT
We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin-a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 10(8) with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.

Show MeSH

Related in: MedlinePlus

Evaluation of candidateaptamers binding to target proteins. (A)Typical F-EMSA results for hHSF1-R5-1 aptamer binding to a two-thirdsdilution series (from 2000 nM to 0.2 nM) of hHSF1 protein. (B, C)Binding curves measured by F-EMSA for hHSF1-R5-1 and hHSF2-R5-2 aptamersto hHSF1, hHSF2, dHSF, and GST tag. The same dilution series in panelA was used in panels B and C. The solid lines are the best fits ofthe Hill equation to the experimental data for each aptamer–targetpair with the appropriate KD values givenin the figure legends.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3753675&req=5

fig5: Evaluation of candidateaptamers binding to target proteins. (A)Typical F-EMSA results for hHSF1-R5-1 aptamer binding to a two-thirdsdilution series (from 2000 nM to 0.2 nM) of hHSF1 protein. (B, C)Binding curves measured by F-EMSA for hHSF1-R5-1 and hHSF2-R5-2 aptamersto hHSF1, hHSF2, dHSF, and GST tag. The same dilution series in panelA was used in panels B and C. The solid lines are the best fits ofthe Hill equation to the experimental data for each aptamer–targetpair with the appropriate KD values givenin the figure legends.

Mentions: The putative RNA aptamers were fluorescentlyend-labeled and thentested for binding to their hHSF targets by F-EMSA and FP assays.16 An image of a typical F-EMSA result is shownin Figure 5A for hHSF1-R5-1 aptamer bindingto hHSF1 protein. The fraction of bound aptamer was calculated asa function of protein concentration and then plotted as shown in Figures 5B and 5C for various aptamer–proteinpairings; KD values were determined byfitting each data set to the Hill equation. Overall, both aptamersshowed high-affinity binding to hHSF1 and hHSF2 (KD < 20 nM). Interestingly, both aptamers also boundto hexahistidine-tagged dHSF, although slightly more weakly (KD ∼ 70 nM), and no binding was observedto the GST-tag alone. The F-EMSA results were confirmed by the FPassays (Figures S-5 and S-6, Supporting Information). Thus, the observed binding is not due to the affinity tags onthe protein targets but rather to specific domains of the targetsthemselves. Given that the highest degree of sequence similarity betweenhHSF1, hHSF2, and dHSF is in the DNA binding and trimerization domains(DBD-TD)26 and that the previously selecteddHSF aptamer was found to bind the DBD-TD of dHSF,21 we predict these novel hHSF aptamers are likely to bindthe HSF proteins in a similar fashion. Contrary to their functionalsimilarity, these two aptamers did not show any similarity in secondarystructure as predicted by mFold27 (FigureS-4, Supporting Information). Althoughbeyond the scope of the present work, the detailed mechanism of theseand other potential aptamers’ binding to HSF proteins as wellas the consequences of binding await further study. However, successfulselection of two distinct high-affinity aptamers, hHSF1-R5-1 and hHSF2-R5-2,targeting two closely related proteins, hHSF1 and hHSF2 respectively,in a single selection demonstrates that our microcolumn-based SELEXtechnology is capable of yielding high-affinity aptamers (KD < 20 nM) in as little as five rounds ofselection, whereas most conventional SELEX methods require typically12 rounds of selection.7


Multiplexed microcolumn-based process for efficient selection of RNA aptamers.

Latulippe DR, Szeto K, Ozer A, Duarte FM, Kelly CV, Pagano JM, White BS, Shalloway D, Lis JT, Craighead HG - Anal. Chem. (2013)

Evaluation of candidateaptamers binding to target proteins. (A)Typical F-EMSA results for hHSF1-R5-1 aptamer binding to a two-thirdsdilution series (from 2000 nM to 0.2 nM) of hHSF1 protein. (B, C)Binding curves measured by F-EMSA for hHSF1-R5-1 and hHSF2-R5-2 aptamersto hHSF1, hHSF2, dHSF, and GST tag. The same dilution series in panelA was used in panels B and C. The solid lines are the best fits ofthe Hill equation to the experimental data for each aptamer–targetpair with the appropriate KD values givenin the figure legends.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3753675&req=5

fig5: Evaluation of candidateaptamers binding to target proteins. (A)Typical F-EMSA results for hHSF1-R5-1 aptamer binding to a two-thirdsdilution series (from 2000 nM to 0.2 nM) of hHSF1 protein. (B, C)Binding curves measured by F-EMSA for hHSF1-R5-1 and hHSF2-R5-2 aptamersto hHSF1, hHSF2, dHSF, and GST tag. The same dilution series in panelA was used in panels B and C. The solid lines are the best fits ofthe Hill equation to the experimental data for each aptamer–targetpair with the appropriate KD values givenin the figure legends.
Mentions: The putative RNA aptamers were fluorescentlyend-labeled and thentested for binding to their hHSF targets by F-EMSA and FP assays.16 An image of a typical F-EMSA result is shownin Figure 5A for hHSF1-R5-1 aptamer bindingto hHSF1 protein. The fraction of bound aptamer was calculated asa function of protein concentration and then plotted as shown in Figures 5B and 5C for various aptamer–proteinpairings; KD values were determined byfitting each data set to the Hill equation. Overall, both aptamersshowed high-affinity binding to hHSF1 and hHSF2 (KD < 20 nM). Interestingly, both aptamers also boundto hexahistidine-tagged dHSF, although slightly more weakly (KD ∼ 70 nM), and no binding was observedto the GST-tag alone. The F-EMSA results were confirmed by the FPassays (Figures S-5 and S-6, Supporting Information). Thus, the observed binding is not due to the affinity tags onthe protein targets but rather to specific domains of the targetsthemselves. Given that the highest degree of sequence similarity betweenhHSF1, hHSF2, and dHSF is in the DNA binding and trimerization domains(DBD-TD)26 and that the previously selecteddHSF aptamer was found to bind the DBD-TD of dHSF,21 we predict these novel hHSF aptamers are likely to bindthe HSF proteins in a similar fashion. Contrary to their functionalsimilarity, these two aptamers did not show any similarity in secondarystructure as predicted by mFold27 (FigureS-4, Supporting Information). Althoughbeyond the scope of the present work, the detailed mechanism of theseand other potential aptamers’ binding to HSF proteins as wellas the consequences of binding await further study. However, successfulselection of two distinct high-affinity aptamers, hHSF1-R5-1 and hHSF2-R5-2,targeting two closely related proteins, hHSF1 and hHSF2 respectively,in a single selection demonstrates that our microcolumn-based SELEXtechnology is capable of yielding high-affinity aptamers (KD < 20 nM) in as little as five rounds ofselection, whereas most conventional SELEX methods require typically12 rounds of selection.7

Bottom Line: We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations.We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2.High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds.

View Article: PubMed Central - PubMed

Affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853, United States.

ABSTRACT
We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin-a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 10(8) with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.

Show MeSH
Related in: MedlinePlus