Chemical RNA Synthesis


Internship Report, 2014
10 Pages, Grade: 1,0
Anonymous

Excerpt

Table of Contents

1 Introduction

2 Material and methods
2.1 Preparations and synthesis
2.2 Cleavage from CPG and deprotection of the base
2.3 RP-HPLC
2.4 DMT cleavage
2.5 Gel analysis

3 Results
3.1 Synthesis
3.2 RP-HPLC
3.3 Gel analysis

4 Discussion
4.1 Synthesis
4.2 RP-HPLC
4.3 DMT cleavage
4.4 Gel analysis

1 Introduction

Oligonucleotides that are used in various experiments such as PCR, Next Generation Sequencing or quantitative PCR can be synthesized chemically. In contrast to the natural 5’→3’ synthesis, the chemical synthesis generates the strand in a 3’→5’ direction. The first base is coupled covalently to Controlled Pore Glass (CPG). The cyclic elongation reaction consists of five steps: detritylation, tetrazolactivation, coupling, capping and oxidation (figure 1). The synthesis is automated by a synthesizing machine.

Here, we exemplarily synthesized an RNA 20mer with and without a 5’-dimethoxytrityl protection group, respectively, in a 0.2 μmol synthesis scale.

illustration not visible in this excerpt

Figure 1: Schematic overview of chemical oligonucleotide synthesis

2 Material and methods

2.1 Preparations and synthesis

The synthesis machine (Applied Biosystems 394 DNA/RNA Synthesizer) was prepared according to the manufacture’s instructions. The phosphoramidites were soluted in acetonitrile, all reagents and columns were installed in the correct position, the capilliaries were flushed and the oligonucleotide sequence (20mer: 5’ CTC ATG GAC GTG CTG GTG AC 3’) was entered. The correcet settings were entered and the synthesis was started. After each cycle, the concentration of the trityl group of the dimethoxyl carbocation was measured automatically to calculate the repetitive yield. It should be over 98% in order to synthesize the oligonucleotide in a sufficient yield, since the repetitive yields multiply. For a 20mer, the minimal total yield could be calculated:

Two syntheses were conducted parallelly, and on- and and off-version, respectively. The on-version contains the final 5’ protecting group, while the off-version does not. This was done to show the importance of the 5’ protecting group for RP-HPLC purification.

2.2 Cleavage from CPG and deprotection of the base

The alkaline cleavage of the synthesized oligonucleotides from the CPG and the deprotection of the base was done with 40% methylamine in H2O for 30 min at 65°C.

To remove the methylamine that disturbs RP-HPLC, a Nap column was used. The column previously had to be equilibrated with ammoniacal H2O. 1 ml of the sample were applied and eluted with 1.5 ml H2O.

2.3 RP-HPLC

RP-HPLC was conducted with a polystyrol column (Resource RP 3 ml, Pharmacia, GE) and a gradient of decreasing hydrophilicity. 50 ml or 500 ml were injected, respectively. The peak fractions were collected manually and dried overnight. Buffer composition and settings are shown below:

illustration not visible in this excerpt

2.4 DMT cleavage

Since not all fractions of the off-oligonucleotide were dry, the dimethyltrytil group was only cleaved from the on product. This was done with 400 ml 80% acetic acid for 30 min at room temperature. The solution was put into the first of four tubes that were used to collect the peak fractions. The tubes were vortexed, centrifuged and the solution was transferred from one tube to another to pool the product. After 30 min, 600 ml H2O were added to get a 1 ml sample. The whole sample was applied on the Nap column and eluted with 1.5 ml H2O to remove the acetic acid.

2.5 Gel analysis

The OD of the samples was measured at l = 260 nm. Different amounts (see next chapter) were mixed with 2x sample buffer and applied on a 12% polyacrylamide gel for analytical and preparative analysis. The gel was run for 1 h at 500 V. The band for preparative analysis should have been cut out with the aid of UV shadowing, the rest of the gel should have been stained with ethidium bromide. The cut out band should have been eluted and the elution fraction should have been applied to the RP-HPLC, again.

Unfortunately, the gel did not show distinct bands, so band was cut out. The whole gel was stained.

[...]

Excerpt out of 10 pages

Details

Title
Chemical RNA Synthesis
College
Free University of Berlin  (Institut für Chemie und Biochemie)
Course
Methodenmodul Nukleinsäuren
Grade
1,0
Year
2014
Pages
10
Catalog Number
V282479
ISBN (eBook)
9783656823421
ISBN (Book)
9783656823438
File size
746 KB
Language
English
Tags
chemical, synthesis
Quote paper
Anonymous, 2014, Chemical RNA Synthesis, Munich, GRIN Verlag, https://www.grin.com/document/282479

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