"In vitro" Selection of rPrPc-binding RNA Molecules

Table of Contents

1 Introduction

2 Material and methods
2.1 RNA preparation
2.2 Protein preparation
2.3 In vitro Selection

3 Results
3.1 RNA preparation
3.1.1 T7 transcription
3.1.1.1 Calculation of pipetting scheme
3.1.1.2 Calculation of DNA template stock concentration
3.1.2 Purification of the transcription product
3.1.2.1 Calculation of RNA copies per template molecule
3.1.2.2 Calculation of purification efficiency
3.1.2.3 Calculation of required RNA amount for desired concentration
3.1.2.4 Calculation of selected RNA amount
3.2 Protein purification
3.2.1 Bradford assay of eluted fractions from Ni-NTA
3.2.2 Analysis of protein purification by SDS-PAGE
3.2.3 Analysis of PCR products by PAGE

4 Discussion
4.1 RNA preparation
4.2 Protein preparation
4.3 In vitro selection

1 Introduction

Aptamers can be considered as nucleic acid based analogues of monoclonar antibodies. These DNA or RNA molecules are characterized by a high affinity and selectivity to the target as well as a huge variety of possible targets, but feature significant advantages compared to antibodies such as the lack of immunogenicity and economic in vitro generation.

The preparation of aptamers is based on systematic evolution (see figure 1). Cycles of selection and replication are conducted to narrow down a huge library to aptamers with the best binding properties to the target.

In this experiment, an RNA aptamer to the rPrPc protein is selected in vitro. Since a whole SELEX procedures takes several weeks, only one selection cycle is done examplarily.

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Figure 1: Principle of SELEX for RNA aptamers

2 Material and methods

The experiments were conducted following the instructions of the script with minor deviations that are recorded in this minutes.

2.1 RNA preparation

Chemical synthesis of DNA templates was done beforehand by the supervisor.
For the T7 transcription, the following pipetting scheme in table 1 was used.

Table 1: Pipetting scheme for T7 transcription (final volume 200 μl)

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After incubation for 2.75 h at 37°C, a 1 μl aliquot was taken and 10 μl DNase I (1 U/μg, RNase-free, Fermentas) was added, followed by another incubation for 15 min at 37°C.

Purification of the T7 transcrption product was performed on a denaturing 12% polyacrylamide gel (figure 2). The gel was pre-run for 15 min at 500-600 V and the samples were mixed with 200 μl urea mixture from the riboyzyme cleavage experiment. The bands of the transcription product were cut out and eluted overnight. The samples of all steps were used for Cerenkov measurements to track the success of purification.

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Figure 2: Denaturing 12% polyacrylamide gel for RNA transcript purification

2.2 Protein preparation

rPrPc (Cricetinae) was overexpressed beforehand by the supervisor in E. coli BL21/DE3 using the pET-15b-vector carrying the PRNP90-231 gen.

All of cells were lysed in a batch. 4 ml MgCl2 were added after stirring for 30 min. Centrifugation was performed in a Sorvall centrifuge at 15,300 g (JA-14).

The frozen pellets were mixed with 50 ml denaturing buffer to resuspend. The suspension was centrifuged in a Sorvall centrifuge at 13,800 g (JA-17).

Purification of rPrPc was achieved by affinity chromatography on Ni-NTA-agarose beads. The beads were prewashed twice with denaturing buffer and spun down for 4-5 min at 3,500 rpm in a Heraeus Labofuge 400. The gradient contains urea from 7 M to 0 M to allow refolding of the protein.

100 ml of the washing buffer (pH 7.0) were prepared and 50 mM imidazole were added. Equally, 100 ml of the elution buffer were prepared at pH 7.0 containing 500 mM imidazole.

The loaded column was washed with 15 ml washing buffer and then with 15 ml 15 ml washing buffer containing 50 mM imidazole to remove unspecific binders.

Eluted fractions were collected in 1.5 ml aliquots and analyzed by Bradford to determine the protein-containing fractions. The results are shown in the next chapter. Fractions 5 to 9 were pooled. To remove imidazole from the sample (“desalting”), the sample was dialyzed overnight in 2 l H2O that contained also 5 mM NaAc (pH 5.5) to stabilize the struture of the protein.

During the protein preparation, aliquots were taken at different steps to track the success of the purification by Bradford. Instead of PBS, H2O was used.

Table 2: Aliquots to track the success of the protein purification

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The protein samples were loaded on a Laemmli SDS-PAGE that was prepared as described below in table 3.

Table 3: Composition of the SDS-PAGE

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2.3In vitroSelection

After purification procedures, the prepared RNA and protein were used for in vitro selection.

RNA molecules were incubated with rPrPc for 45 min. Protein-bound RNA molecules were selected on a nitrocellulose membrane. In order to do so, the membrane was wetted with H2O, loaded with the sample and washed four times with 500 μl 1x binding buffer (2 ml in total) to remove RNA that was not bound to the target protein. Elution from the filter was performed twice with 300 μl 7M urea for 15 min at 90°C. The eluates were pooled.

Chloroform/phenol extraction of the RNA was performed with 500 μl Chloroform/phenol solution. Cerenkov measurement of several purification steps was performed (see next chapter) to determine the yield of bound RNA molecules.

500 μl RNA were mixed with 1 ml EtOH and 1 μl glycogen to precipitate the nucleic acids at -70°C overnight. The precipitation mixture was centrifuged for 1 h at 4°C and 13,000 rpm (15,700 g, eppendorf centrifuge 5415 R) to pelletize the precipitated RNA. The supernatant was discarded and the pellet was dried in a SpeedVac (vacuubrand).

In order to amplify binding sequences with rPrPc affinity, reverse transcription and PCR were performed.

Reverse transcription was prepared according to the following scheme in table 4.

Table 4: Pipetting scheme for reverse transcription (final volume 20 μl)

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Table 5: Pipetting scheme for PCR (final volume 100 μl)

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PCR products were analyzed on a 12% polyacrylamide gel. 10 μl of each PCR sample were mixed with 10 μl loading buffer. The DNA library served as positive control. 3 μl of the DNA library were mixed with 5 μl loading buffer. The gel was stained with etidium bromide.

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