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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.

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Aptamerselection workflow for multiple targets by use of microcolumns.(A) Microcolumn filled with 10 μL of GFP-immobilized chromatographyresin. (B) Multiplexed selection of RNA aptamers: (1) The startingRNA library is dynamically loaded onto multiple microcolumns thatare connected in a serial configuration. (2) The devices are rearrangedinto a parallel configuration and the subsequent cycles in the processare done independently but simultaneously. (3) Unbound and weaklybound RNAs are washed away. (4) The remaining bound RNAs are elutedfrom each column separately. (5) The RNA molecules are reverse-transcribedinto cDNA and (6) a small fraction is analyzed via qPCR. (7) The remainingcDNA is PCR-amplified and then (8) transcribed back into RNA to makea new amplified pool for (9) the next selection round. The steps shownwith dashed arrows are optional and are not necessarily done in eachround.
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fig1: Aptamerselection workflow for multiple targets by use of microcolumns.(A) Microcolumn filled with 10 μL of GFP-immobilized chromatographyresin. (B) Multiplexed selection of RNA aptamers: (1) The startingRNA library is dynamically loaded onto multiple microcolumns thatare connected in a serial configuration. (2) The devices are rearrangedinto a parallel configuration and the subsequent cycles in the processare done independently but simultaneously. (3) Unbound and weaklybound RNAs are washed away. (4) The remaining bound RNAs are elutedfrom each column separately. (5) The RNA molecules are reverse-transcribedinto cDNA and (6) a small fraction is analyzed via qPCR. (7) The remainingcDNA is PCR-amplified and then (8) transcribed back into RNA to makea new amplified pool for (9) the next selection round. The steps shownwith dashed arrows are optional and are not necessarily done in eachround.

Mentions: To address these limitations, we developed a processutilizingreconfigurable microcolumns of varying capacity for selecting RNAaptamers. The microcolumns require only microliter volumes of affinitychromatography resin (∼2–50 μL), they can be easilyassembled in various configurations to accommodate multiple targets,and they can be easily integrated with common laboratory equipment.In addition, these microcolumns are not restricted to RNA and othernucleic acid aptamer selections but are also suitable for other affinitychromatography needs where small column volumes are desired. The assemblyof microcolumns and aptamer selection process are shown in Figure 1 with the experimental details provided below.


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)

Aptamerselection workflow for multiple targets by use of microcolumns.(A) Microcolumn filled with 10 μL of GFP-immobilized chromatographyresin. (B) Multiplexed selection of RNA aptamers: (1) The startingRNA library is dynamically loaded onto multiple microcolumns thatare connected in a serial configuration. (2) The devices are rearrangedinto a parallel configuration and the subsequent cycles in the processare done independently but simultaneously. (3) Unbound and weaklybound RNAs are washed away. (4) The remaining bound RNAs are elutedfrom each column separately. (5) The RNA molecules are reverse-transcribedinto cDNA and (6) a small fraction is analyzed via qPCR. (7) The remainingcDNA is PCR-amplified and then (8) transcribed back into RNA to makea new amplified pool for (9) the next selection round. The steps shownwith dashed arrows are optional and are not necessarily done in eachround.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Aptamerselection workflow for multiple targets by use of microcolumns.(A) Microcolumn filled with 10 μL of GFP-immobilized chromatographyresin. (B) Multiplexed selection of RNA aptamers: (1) The startingRNA library is dynamically loaded onto multiple microcolumns thatare connected in a serial configuration. (2) The devices are rearrangedinto a parallel configuration and the subsequent cycles in the processare done independently but simultaneously. (3) Unbound and weaklybound RNAs are washed away. (4) The remaining bound RNAs are elutedfrom each column separately. (5) The RNA molecules are reverse-transcribedinto cDNA and (6) a small fraction is analyzed via qPCR. (7) The remainingcDNA is PCR-amplified and then (8) transcribed back into RNA to makea new amplified pool for (9) the next selection round. The steps shownwith dashed arrows are optional and are not necessarily done in eachround.
Mentions: To address these limitations, we developed a processutilizingreconfigurable microcolumns of varying capacity for selecting RNAaptamers. The microcolumns require only microliter volumes of affinitychromatography resin (∼2–50 μL), they can be easilyassembled in various configurations to accommodate multiple targets,and they can be easily integrated with common laboratory equipment.In addition, these microcolumns are not restricted to RNA and othernucleic acid aptamer selections but are also suitable for other affinitychromatography needs where small column volumes are desired. The assemblyof microcolumns and aptamer selection process are shown in Figure 1 with the experimental details provided below.

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