TITLE: Plate cell – SELEX
AUTHORS: Claudia Troncone Clemente

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Aptamers are short single-stranded oligonucleotide (DNA or RNA) molecules with the ability to bind to other molecules with high affinity and specificity. Nowadays, aptamers are used as biosensing molecules with broader applications, such as ribozymes (RNA enzyme molecules with catalytic activities); riboswitches (modulating translational activities) and ligands, recognizing specific target binding. Aptamers are folded, directed by their sequences, acquiring specific 3D conformations. This 3D conformation property has been widely applied in order to recognize cell structures.</div><div class = "text-block"><span>Aptamers evolve from random oligonucleotide pools by a process called </span><span style = "font-weight:bold;">Systematic Evolution of Ligands by Exponential enrichment (SELEX)</span><span>. Conceptually, the SELEX process is controlled by the ability of these small oligonucleotides to fold into unique 3D structures that can interact with a specific target with high specificity and affinity [1]. This is a dynamic process, repeating exposures and elutions, allowing to screen in a heterogenic pool in low concentration ranges.</span></div><div class = "text-block">A standard SELEX method has four major steps: </div><div class = "text-block">    1.- Exposure of random sequence single stranded nucleotide oligo library to a target. </div><div class = "text-block">    2.- Binding of oligos to the target molecule. </div><div class = "text-block">    3.- Selection of binders and removal of non-binding oligos.</div><div class = "text-block">    4.- Amplification of the binder fraction and portioning of the amplicon to single strand. </div><div class = "text-block">These steps are iteratively performed till a pool of high binding aptamer is screened out from the library. The oligonucleotide library consists of a random base-sequence flanked on both ends by primer binding sites, which aids in amplification and enrichment (Safeh et al., 2010; Kim et al., 2013).</div><div class = "text-block">Here is described how SELEX have been applied to develop aptamers, recognizing specific outer membrane structures, as LamB.</div></div>

[STEPS]
?. [DAY 1]
Preparation of the inoculum
?. [DAY 1]
Microtiter plate functionalization
?. [DAY 2]
Growing and harvesting cells
?. [DAY 2]
SELEX INITIAL ROUND: Exposure and Screening
?. [DAY 1]
In 1mL of LB liquid medium, resuspend the E. coli cells.Incubate it at 37 °C overnight with constant shaking at 180 rpm in a Incubator-shaker
?. [DAY 1]
Take a sterile microtiter plate and coat it with 100 μl of 2.5 mM PBA prepared in carbonate buffer (pH 9.2).Incubate the plate overnight at 4ºC. This will make the well surface to functionalize with PBA.
?. [DAY 1]
Strcuturalization
?. [DAY 1]
Add 10 μL of 100 uM ssDNA library to 190 ul of binding buffer. Incubate it at 95 °C for 10 min. After that, transfer immediately to ice-bath to prevent rehybridization ofthe single stranded DNA library. If the transfer is poor performed, you can reheat again for another 10 min and repeat.Incubate the tube at 4 °C  overnight (in the refrigerator). This will allow the single stranded DNA oligos to taketheir respective 3D conformations.
?. [DAY 2]
With the spectrophotometer set at λ = 600 nm, measure the growth of the inoculum. It should be ≥ 1. If it's lower, it should be left growing until it reaches this value.
?. [DAY 2]
Take 250 μL of bacteria and inoculate it in 25 ml LB media.(use 1% O.D. as an inoculum for subculturing)
?. [DAY 2]
Incubate at 37ºC, 180 rpm for 1.5h (until the culture reaches an O.D. between 0.5 – 0.6, which will indicate that the culture is in log-phase)
?. [DAY 2]
Centrifuge the tube at 3000 X g for 10 minutes
?. [DAY 2]
Remove supernatant and then wash the cells x3 with PBS (10 mM, pH 7.4). This will remove residual components from the previous step.
?. [DAY 2]
Measure the O.D. at =600 nm using a spectrophotometer at λ = 600 nm. The optical density (O.D.) shouldbe ≥ 1. This corresponds approximately to 1 x 108 E. coli cells (diluted in PBS). Further dilutions can be done by adding PBS to tubes.
?. [DAY 2]
Wash the PBA coated plate x2 with carbonate buffer. Then, resuspend 100 μL of 1 x 103 cells in well #1.
?. [DAY 2]
Incubate at 25 ºC for 1h. This will allow the cells to coat the surface of the well
?. [DAY 2]
Decant the suspension by inversion and wash the well with 200 μL of Binding buffer (x3). The washing will remove the cells that didn't bind to the surface.
?. [DAY 2]
Incubate 200 μL of naïve library (prepared the previous day) at 37ºC for 15 minutes
?. [DAY 2]
Add 200 μL of library to the cell coated well (gently!) and then incubate at 37ºC for 1h.
?. [DAY 2]
Decant the solution from the plate by inverting.
?. [DAY 2]
Wash x3 with 200 ul of Binding buffer to separate loosely bound sequences
?. [DAY 2]
Add 200 μL of elution buffer to the well and then incubate at room temperature for 5 min.
?. [DAY 2]
Remove the solution by using micropipette (be careful not to touch the walls) and neutralize inmediately by adding 10 μL of neutralization buffer. This will bring the pH to ~7.
?. [DAY 2]
Quantify the obtained sequences with the spectrophotometer and label as Round #1.To avoid bacterial contamination due to detachment, the DNA elutes can be centrifuged at 3,000 x g for 10 min and take the supernatant prior to quantification. This can be stored at -20 °C till further use.