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Molecular and Functional Characterization of ssDNA Aptamers that Specifically Bind Leishmania infantum PABP.

Guerra-Pérez N, Ramos E, García-Hernández M, Pinto C, Soto M, Martín ME, González VM - PLoS ONE (2015)

Bottom Line: Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population.Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity.At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzed.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of aptamers, Servicio de Bioquímica-Investigación, IRYCIS-Hospital Ramón y Cajal, Madrid, Spain.

ABSTRACT

Summary: A poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study.

Results: We have selected a ssDNA aptamer population against a recombinant 6xHIS-LiPABP protein (rLiPABP) that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP) protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A) in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes.

Conclusion: Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzed.

No MeSH data available.


Related in: MedlinePlus

Aptamers are selected after 4 round of SELEX.The stringency of the SELEX procedure was monitored using ELONA as described in “Materials and Methods” section. SELEX pool from round 4 (SELLiPABP) and the starting round (RND40) were assayed for binding to LiPABP protein. Recombinant LiPABP was plated at 500 ng/well (7.5 pmol/well) and incubated with 200 μL of digoxigenin labeled SELLiPABP aptamer population or digoxigenin-labeled RND40 library (0–80 nM). Finally, anti-digoxigenin-POD antibodies were added plate was revealed with ABTS solution at 405 nm. All the experiments were made in triplicate and average of two different experiments is shown.
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pone.0140048.g001: Aptamers are selected after 4 round of SELEX.The stringency of the SELEX procedure was monitored using ELONA as described in “Materials and Methods” section. SELEX pool from round 4 (SELLiPABP) and the starting round (RND40) were assayed for binding to LiPABP protein. Recombinant LiPABP was plated at 500 ng/well (7.5 pmol/well) and incubated with 200 μL of digoxigenin labeled SELLiPABP aptamer population or digoxigenin-labeled RND40 library (0–80 nM). Finally, anti-digoxigenin-POD antibodies were added plate was revealed with ABTS solution at 405 nm. All the experiments were made in triplicate and average of two different experiments is shown.

Mentions: The initial RND40 population and the selected SELLiPABP pool were then tested for its ability to bind rLiPABP using ELONA. In only four rounds of aptamer selection, SELLiPABP population displayed increased binding to rLiPABP protein compared with the initial pool suggesting that it is enriched in ssDNA sequences that recognize rLiPABP relative to the naïve RND40 population (Fig 1). Consequently, we decided to analyze the binding affinity of the SELLiPABP aptamer population to L. infantum LiPABP using ELONA and slot blot assays. Thus, in a first approach, the wells were coated with recombinant LiPABP protein and different concentrations of digoxigenin-labeled SELLiPABP were tested as described in Materials and methods section (Fig 2A). Data were analyzed using non-linear regression showing that they responds to a one-site binding curve with an equation y = (x × Bmax)/(x + Kd) where Bmax is the maximal binding and Kd is the concentration of ligand required to reach half-maximal binding. Results indicate that SELLiPABP is able to detect rLiPABP in a concentration-dependent manner with Kd = 3.87 ± 0.67 nM. In addition, an exhaustive analysis of the data represented in Fig 2A showed that a concentration so low as 2.48 nM of SELLiPABP is able to detect from 500 ng (7.5 pmol) of recombinant LiPABP in a significant manner (p<0.01).


Molecular and Functional Characterization of ssDNA Aptamers that Specifically Bind Leishmania infantum PABP.

Guerra-Pérez N, Ramos E, García-Hernández M, Pinto C, Soto M, Martín ME, González VM - PLoS ONE (2015)

Aptamers are selected after 4 round of SELEX.The stringency of the SELEX procedure was monitored using ELONA as described in “Materials and Methods” section. SELEX pool from round 4 (SELLiPABP) and the starting round (RND40) were assayed for binding to LiPABP protein. Recombinant LiPABP was plated at 500 ng/well (7.5 pmol/well) and incubated with 200 μL of digoxigenin labeled SELLiPABP aptamer population or digoxigenin-labeled RND40 library (0–80 nM). Finally, anti-digoxigenin-POD antibodies were added plate was revealed with ABTS solution at 405 nm. All the experiments were made in triplicate and average of two different experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4601788&req=5

pone.0140048.g001: Aptamers are selected after 4 round of SELEX.The stringency of the SELEX procedure was monitored using ELONA as described in “Materials and Methods” section. SELEX pool from round 4 (SELLiPABP) and the starting round (RND40) were assayed for binding to LiPABP protein. Recombinant LiPABP was plated at 500 ng/well (7.5 pmol/well) and incubated with 200 μL of digoxigenin labeled SELLiPABP aptamer population or digoxigenin-labeled RND40 library (0–80 nM). Finally, anti-digoxigenin-POD antibodies were added plate was revealed with ABTS solution at 405 nm. All the experiments were made in triplicate and average of two different experiments is shown.
Mentions: The initial RND40 population and the selected SELLiPABP pool were then tested for its ability to bind rLiPABP using ELONA. In only four rounds of aptamer selection, SELLiPABP population displayed increased binding to rLiPABP protein compared with the initial pool suggesting that it is enriched in ssDNA sequences that recognize rLiPABP relative to the naïve RND40 population (Fig 1). Consequently, we decided to analyze the binding affinity of the SELLiPABP aptamer population to L. infantum LiPABP using ELONA and slot blot assays. Thus, in a first approach, the wells were coated with recombinant LiPABP protein and different concentrations of digoxigenin-labeled SELLiPABP were tested as described in Materials and methods section (Fig 2A). Data were analyzed using non-linear regression showing that they responds to a one-site binding curve with an equation y = (x × Bmax)/(x + Kd) where Bmax is the maximal binding and Kd is the concentration of ligand required to reach half-maximal binding. Results indicate that SELLiPABP is able to detect rLiPABP in a concentration-dependent manner with Kd = 3.87 ± 0.67 nM. In addition, an exhaustive analysis of the data represented in Fig 2A showed that a concentration so low as 2.48 nM of SELLiPABP is able to detect from 500 ng (7.5 pmol) of recombinant LiPABP in a significant manner (p<0.01).

Bottom Line: Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population.Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity.At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzed.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of aptamers, Servicio de Bioquímica-Investigación, IRYCIS-Hospital Ramón y Cajal, Madrid, Spain.

ABSTRACT

Summary: A poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study.

Results: We have selected a ssDNA aptamer population against a recombinant 6xHIS-LiPABP protein (rLiPABP) that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP) protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A) in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes.

Conclusion: Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzed.

No MeSH data available.


Related in: MedlinePlus