Difference between revisions of "Part:BBa K1636000"

Revision as of 23:45, 26 September 2015

Lead-II-ions-specific aptamer

This aptameric sequence is capable of recognising and binding specifically to lead II ions. This sequence, which works as a single stranded nucleic acid molecule, is capable of forming a G-quadruplex by interacting with lead ions (Pb2+) while disrupting a partially complementary duplex.

Sequence and Features

The linker was ligated into the linearised plasmid pSB1C3 and previously cut with EcoRI and PstI, then a purification was performed (figure 1A). Several clones were selected to isolate plasmid by miniprep (figure 1). In order to analyse the integrity of plasmids an electrophoresis was run (figure 1B). Several plasmid isoforms were observed and the plasmids were suitable to be used as template in a PCR reaction to know the presence of the insert.

Figure 1. A) Scheme of aptamer cloning in pSB1C3 plasmid and B) electrophoresis with agarose gel at 0.8% to analyse plasmid extraction from different clones (lanes 2 and 3 with 10 microliters of each sample + 4 microliters of loading buffer).

To know if the aptamer was cloned in pSB1C3 a PCR was performed using universal primers VF2 and VR (figure 2A). Those primers amplify a PCR product of 299 pb when the insert is present (figure 1A). Plasmids from 2 different clones were tested and one of them had the PCR product of approximately 299 bp corresponding to the plasmid with insert (figure 2B, lane 3).

Figure 2. Analysis of the aptamer cloning into pSB1C3 plasmid by PCR. A) Plasmid scheme with the position of the cloned aptamer wich is flanked by the restriction sites EcoRI and PstI, also the alignment position of primers VF2 and VR is indicated. B) Plasmids from 2 different clones were used as templates for PCR with universal primers VF2 and V (lanes 2-3). Lane 1 contains Quick-Load® 2-Log DNA Ladder(0.1-10.0 kb), NEB.

Measurement of aptamer fluorescence by ZnPPIX and Pb(NO3)2

This essay was performed in order to check the ability that certain oligonucleotides have of capturing lead, including the aptamer (K1636000), the aptamer flanked by restriction enzymes sites EcoRI and PstI, scaffold 3 and DNAbot. The protocol used was based on the report of Li et al 2010.

Protocol based on Li et al, 2010 with several modifications described below: Reactions with 200 ul as final volume was prepared as following: - 6 uM of oligonucleotides 1 or 2 or 10 nM of oligonucleotides 3 or 4 - 10mM Tris-Ac buffer, 10 uM ZnPPIX - 50mM MOPS buffer, 10uM Pb(NO3)2.

Samples were transferred to a white opaque plate. Fluorescences were read at different times for 2 hours (time used in Li et al 2010) using a plate reader Synergy-H4 with an excitation wavelength of 420 nm and fluorescence emission at 600 nm. A set of tests were performed adding a previous temperature treatment for oligonucleotides consisting on keeping samples at 80°C for 10 min in a water bath, followed by slightly cooling down at room temperature and the addition of ZnPPIX, MOPS buffer and Pb(NO3)2. All reagents, except oligonucleotides 3 and 4, were in the same concentrations than the ones reported in the experiments of Li et al 2010.

Figure 3 depicts the fluorescence emission of oligonucleotides and some prepared blanks.The assay was based upon the reported protocol by Li et al un 2010. However, we were not able to perform it exactly as he suggested it due to the lack of a reagent: hydroxylamine (NH2OH). This first reaction was carried out in order to determine which components of it contributed to the fluorescence on the assay. Herein, the explanation of each reaction: in blue, the aptamer as reported by Li (i.e. without restriction enzyme sites) as a positive control given that it was already reported as functional, in red our aptamer sequence submitted as BioBrick (i.e. containing additional base pairs for restriction enzyme sites), in green the first negative control for the aptamer just as reported by Li (without restriction enzyme sites) containing all reagents except lead, in purple the negative control for the aptamer BioBrick sequence (with restriction enzyme sites) containing all reagents but lead, in light blue the negative control containing all reagents but DNA molecules and HNO3, and the one in orange is the negative control containing all reagents but lead and HNO3. In this context, we observed that lead solution in nitric acid was a factor that contributed to the emission of fluorescence. We attribute this to the fact that we lacked NH2OH in our assay, for it is a base and probably it helps to counteract the charges of the HNO3. This could have interfered with the electron resonance of the Zinc protoporphyrin thereby enhancing its fluorescence despite the lack of lead in the solution. Therefore, we chose as final blank the reaction without Pb-HNO3. Moreover, this blank was also chosen since it was more stable than the others tested. Figure 3 shows that absence of Pb solution changed dramatically the fluorescence emission. This blank was used to correct the RFU of reactions, Figures 4 and 5 depict corrected RFU of reactions.

Figure 3. Fluorescence emission of oligonucleotides and blanks.

Once the contribution of the reagents was determined, we performed the reaction with the temperature treatment suggested by Li; knowing that the selected blank fluoresced about to 11,000 RFUs, we didn't include its measurements in the graph constructed with the new values for the aptamer sequence (Li) in blue, our aptamer biobrick sequence (red), scaffold 1 (in green), and "assembled" DNAbot in purple. Figure 4 depicts the fluorescence emission of reactions with oligonucleotides pretreated by temperature. The aptamer emitted high fluorescence independently of having or not the restriction enzyme sites because the baseline, determined in the first fluorescence reaction (shown in Figure 3), stabilises about to 11,000 RFUs after two hours which is the time suggested to achieve duplex-quadruplex conversion. The scaffold and DNAbot emitted less fluorescence than the blank. In general, it was observed that the fluorescence emission decreased over time, and stabilised about to two hours after the preparation of the samples.

Figure 4. Fluorescence emission of oligonucleotides with temperature treatment.

Figure 5. Fluorescence emission of oligonucleotides with temperature treatment after 20 h.

As the values changed over time we decided to measure the fluorescence after a longer time span. We measured after 20 hours and at this time the values were more constant. Figure 7 depicts the average of three different measurements. Aptamer with and without restriction enzymes values were very similar, and lower compared to the ones measured at 2 hours time. The scaffold didn’t differ very much from the values taken at 2 hours, but the DNAbot increased significantly, reaching a positive value.

Due to time issues, we have not been able to determine concentration efficiency of the aptamer (i.e. how much lead can be captured by the DNAbot); however, Li has reported that the aptamer is as sensitive as 20 nM of lead in a 6 micromolar concentration of DNA molecules. Given that our DNAbot structured contains 6 aptamers (one per monomer), we expect to achieve higher capturing yield per quantity of DNA nanoparticle. Moreover, we still need to find the optimal assembly protocol for the DNAbot in order for it to undergo the characterisation analysis for the lead-capturing - DNAbot concentration and lead concentration measurement.