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Single-Round Patterned DNA Library Microarray Aptamer Lead Identification.

Martin JA, Mirau PA, Chushak Y, Chávez JL, Naik RR, Hagen JA, Kelley-Loughnane N - J Anal Methods Chem (2015)

Bottom Line: The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation.This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach.This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

View Article: PubMed Central - PubMed

Affiliation: Human Effectiveness Directorate, 711 Human Performance Wing, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, OH 45433, USA ; The Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720A Rockledge Drive, Bethesda, MD 20817, USA.

ABSTRACT
A method for identifying an aptamer in a single round was developed using custom DNA microarrays containing computationally derived patterned libraries incorporating no information on the sequences of previously reported thrombin binding aptamers. The DNA library was specifically designed to increase the probability of binding by enhancing structural complexity in a sequence-space confined environment, much like generating lead compounds in a combinatorial drug screening library. The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation. We propose that the lack of G-quartet structure in microarray-derived aptamers may highlight differences in binding mechanisms between surface-immobilized and solution based strategies. This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach. This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

No MeSH data available.


Microarray performance of the top 15 ranked potential thrombin binders of PT2 compared to controls. Inset: close-up view of the intensities of the top 5 ranked sequences compared to the negative control. Error bars represent standard deviation of replicates of fluorescence values obtained from a 2 h incubation of 100 nM Cy3-thrombin with the microarray at 20°C.
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fig1: Microarray performance of the top 15 ranked potential thrombin binders of PT2 compared to controls. Inset: close-up view of the intensities of the top 5 ranked sequences compared to the negative control. Error bars represent standard deviation of replicates of fluorescence values obtained from a 2 h incubation of 100 nM Cy3-thrombin with the microarray at 20°C.

Mentions: A full description of pattern design and analysis is included in Supporting Information text and Figure S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/137489. The pattern PT2 was applied to the microarray for assessing thrombin binding. Several potential thrombin aptamer candidates were identified from the microarray work. The top 15 candidates were ranked by fluorescence intensity (Figure 1), in comparison to the positive controls of the reported thrombin fibrinogen binding site aptamer (TFBS) and thrombin heparin binding site aptamer (THBS) [21, 22]. A mutation of the streptavidin aptamer (SA) with an extra guanine base inserted at position 19 was used as a negative control due to its low binding observed with a variety of different targets in preliminary work [23]. All of the top 15 reported sequences demonstrated over 12x higher mean fluorescence intensity values than SA, with the highest intensity sequence, 4A018, reporting mean values over 120x higher than SA. All fluorescence intensity values for the top 15 sequences were statistically significant compared to the SA control (p < 0.0001, 95% CI). A close-up of the fluorescence intensity values of the top 5 sequences compared to SA is also shown (Figure 1 inset).


Single-Round Patterned DNA Library Microarray Aptamer Lead Identification.

Martin JA, Mirau PA, Chushak Y, Chávez JL, Naik RR, Hagen JA, Kelley-Loughnane N - J Anal Methods Chem (2015)

Microarray performance of the top 15 ranked potential thrombin binders of PT2 compared to controls. Inset: close-up view of the intensities of the top 5 ranked sequences compared to the negative control. Error bars represent standard deviation of replicates of fluorescence values obtained from a 2 h incubation of 100 nM Cy3-thrombin with the microarray at 20°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4446497&req=5

fig1: Microarray performance of the top 15 ranked potential thrombin binders of PT2 compared to controls. Inset: close-up view of the intensities of the top 5 ranked sequences compared to the negative control. Error bars represent standard deviation of replicates of fluorescence values obtained from a 2 h incubation of 100 nM Cy3-thrombin with the microarray at 20°C.
Mentions: A full description of pattern design and analysis is included in Supporting Information text and Figure S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/137489. The pattern PT2 was applied to the microarray for assessing thrombin binding. Several potential thrombin aptamer candidates were identified from the microarray work. The top 15 candidates were ranked by fluorescence intensity (Figure 1), in comparison to the positive controls of the reported thrombin fibrinogen binding site aptamer (TFBS) and thrombin heparin binding site aptamer (THBS) [21, 22]. A mutation of the streptavidin aptamer (SA) with an extra guanine base inserted at position 19 was used as a negative control due to its low binding observed with a variety of different targets in preliminary work [23]. All of the top 15 reported sequences demonstrated over 12x higher mean fluorescence intensity values than SA, with the highest intensity sequence, 4A018, reporting mean values over 120x higher than SA. All fluorescence intensity values for the top 15 sequences were statistically significant compared to the SA control (p < 0.0001, 95% CI). A close-up of the fluorescence intensity values of the top 5 sequences compared to SA is also shown (Figure 1 inset).

Bottom Line: The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation.This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach.This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

View Article: PubMed Central - PubMed

Affiliation: Human Effectiveness Directorate, 711 Human Performance Wing, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, OH 45433, USA ; The Henry M. Jackson Foundation for the Advancement of Military Medicine, 6720A Rockledge Drive, Bethesda, MD 20817, USA.

ABSTRACT
A method for identifying an aptamer in a single round was developed using custom DNA microarrays containing computationally derived patterned libraries incorporating no information on the sequences of previously reported thrombin binding aptamers. The DNA library was specifically designed to increase the probability of binding by enhancing structural complexity in a sequence-space confined environment, much like generating lead compounds in a combinatorial drug screening library. The sequence demonstrating the highest fluorescence intensity upon target addition was confirmed to bind the target molecule thrombin with specificity by surface plasmon resonance, and a novel imino proton NMR/2D NOESY combination was used to screen the structure for G-quartet formation. We propose that the lack of G-quartet structure in microarray-derived aptamers may highlight differences in binding mechanisms between surface-immobilized and solution based strategies. This proof-of-principle study highlights the use of a computational driven methodology to create a DNA library rather than a SELEX based approach. This work is beneficial to the biosensor field where aptamers selected by solution based evolution have proven challenging to retain binding function when immobilized on a surface.

No MeSH data available.