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.
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
Research Objectives and Focus
The primary objective of this experimental work is to perform an in vitro selection process to identify RNA aptamers that exhibit specific binding properties to the rPrPc protein. By conducting a single selection cycle, the study aims to demonstrate the practical application of the SELEX procedure in isolating target-specific nucleic acid molecules.
- Systematic evolution and in vitro selection of RNA aptamers.
- Methodical preparation and purification of RNA transcripts.
- Expression and purification of recombinant PrPc protein using affinity chromatography.
- Qualitative analysis of selection success via SDS-PAGE and denaturing PAGE.
Excerpt from the book
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.
Summary of Chapters
1 Introduction: This chapter introduces the concept of aptamers as nucleic acid analogues to antibodies and outlines the SELEX principle for their isolation.
2 Material and methods: This section details the experimental protocols, including the preparation and purification of RNA and rPrPc protein, as well as the specific steps taken for the in vitro selection process.
3 Results: This chapter presents the data gathered from transcription, protein purification, and the final selection cycle, supported by quantitative calculations and gel electrophoresis analysis.
4 Discussion: The final section evaluates the success of the experimental procedures, addresses potential bottlenecks such as purification yields, and interprets the qualitative outcomes of the selection.
Keywords
Aptamers, SELEX, rPrPc, RNA selection, In vitro evolution, Protein purification, Ni-NTA, SDS-PAGE, Denaturing PAGE, Transcription, Binding affinity, Nucleic acid biochemistry, Molecular biology, Chromatography, Target specificity.
Frequently Asked Questions
What is the primary focus of this research?
The study focuses on the in vitro selection of RNA molecules capable of binding to the rPrPc protein, utilizing the SELEX methodology.
What are the core thematic areas?
The core themes include RNA aptamer generation, recombinant protein expression and purification, and the verification of binding sequences through electrophoretic techniques.
What is the main goal of the experiment?
The primary goal is to perform a single cycle of the SELEX procedure to isolate and identify RNA sequences that demonstrate binding affinity to the target protein.
Which scientific methods were employed?
The study utilized T7 transcription, affinity chromatography with Ni-NTA-agarose beads, Cerenkov measurements for yield tracking, SDS-PAGE for protein analysis, and denaturing PAGE for nucleic acid analysis.
What is covered in the main section?
The main section covers the detailed protocols for RNA and protein preparation, the specific steps of the in vitro selection, and the subsequent analysis of the results using various measurement and visualization techniques.
Which keywords define this work?
Key terms include aptamers, SELEX, rPrPc, in vitro selection, transcription, protein purification, and PAGE analysis.
Why was only one selection cycle performed?
A full SELEX procedure typically spans several weeks; therefore, this experiment was designed to demonstrate the process through a single, exemplary selection cycle.
How was the success of the protein purification verified?
The purification success was monitored by tracking protein-containing fractions via Bradford assays and visualized through Coomassie Blue-stained SDS-PAGE.
What challenges were noted during the RNA purification?
Challenges included incomplete transcription efficiency and technical difficulties in eluting the RNA from the gel due to the large volume and distribution of the gel pieces.
What can be concluded from the PAGE analysis of the PCR products?
The analysis is qualitative and confirms that target-binding RNA sequences were successfully selected; however, it does not provide quantitative data due to varying replication efficiencies during PCR.
- Quote paper
- Anonym (Author), 2014, "In vitro" Selection of rPrPc-binding RNA Molecules, Munich, GRIN Verlag, https://www.hausarbeiten.de/document/282478
