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Assignment #1: 5 Properties
1. 0.786 g/mL
2. 0.786 g/mL
3. 0.786 g/mL
5. 0.747 g/mL
6. 0.982 g/mL at 20 C
1. -45 °C
2. -48 °C
3. -48 °C
4. -48 °C to -45 °C
5. -48 °C
1. 82 °C
2. 81-82 °C
3. 81.5 °C
4. 80 °C to 82 °C
5. 80 °C to 82 °C
1. 2 °C
2. 5.556 °C
3. 5 °C (41 °F)
4. 5.556 °C
5. 48 °F
Assignment #2: Summary of Article
Title: Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA
Reference: Sean M. McCarthy, Martin Gilar, John Gebler. Analytical Biochemistry 390 (2009) 181-188.
[Full Marks JCB]
Ribonucleic acid interference (RNAi) mechanism for regulation of gene expression was first discovered in plants. The mechanism was later shown to work in worms and mammals. It uses two forms of small RNA molecules: microRNA and small interfering RNA (siRNA). The siRNA are short oligonucleotide sequences (21-23 mer) in double stranded form.
The siRNA molecules for therapeutic use are prepared synthetically. The synthesis process is typically very efficient with high yields and purity of about 90%. Most impurities formed during the synthesis process are from failed sequences, which can cause off-target gene silencing. siRNA are currently studied for therapeutic use in treating different diseases, such as cancer and other viral infections. However, one of the major challenges in developing the siRNA molecules as therapeutics is obtaining good purity.
The gene silencing experiments are usually performed with double stranded siRNA. They are prepared by hybridization of two complementary single stranded siRNA sequences. In this approach, however, each single strand can introduce its own impurities that can result in mismatched sequences and other impurities that can further complicate the double stranded siRNA. The presence of these impurities can also lead to unwanted and non-targeted gene silencing. As a result, efficient analysis and purification approaches for siRNA are very important when developing them for therapeutic use.
Numerous different methods for the analysis and purification of oligonucleotides have been reported. These include polyacrylamide gel electrophoresis (PAGE), capillary gel electrophoresis (CGE), anion-exchange high-performance liquid chromatography (AX-HPLC) and ion-pair liquid chromatography (IP-LC).
IP-RPLC method using triethylamine and hexafluoroisopropanol (HFIP) as ion pairing reagents provides good resolution for the separation of oligonucleotides and are compatible with mass spectrometry (MS).
The double stranded siRNA are typically prepared by separately purifying each complementary single strand and then annealing the two strands to form the siRNA. In this article, the authors propose a different approach for the purification of double stranded siRNA where the crude complementary single stranded siRNA are annealed on-column to form the double stranded siRNA.
In the materials section, the authors list all materials used in the studies and where they were purchased.
In the methods section, detailed information about method information and parameters are listed.
The IP-RPLC method was used for the purification of siRNA. Using this method, up to 140 nmol of 21-mer siRNA was purified in a single injection using analytical size column. However, as the sample load increased on the column, the typical peak broadening for overloaded conditions was observed. The sample was collected using the "heart cutting" approach in preparative chromatography with minimum product loss.
About 1-2 mL fractions were collected for each injection on the column. The yield was between 55% and 70% based on the peak area. Further UPLC analysis of the collected fractions showed >95% purity.
siRNA can be purified in single-stranded or double stranded form. The latter approach is typically more efficient, but a few problems need to be considered. One of the major concerns is the siRNA duplex stability under the IP-RPLC conditions. The melting temperatures of duplex siRNA are between 45 and 65 C, and on-column melting can potentially occur during the purification process.
In order to investigate the stability of duplex siRNA under IP-RPLC conditions, the authors chose three different duplexes that were prepared from purified single-stranded counterparts and with melting temperatures of 47.5 C, 57.0 C, and 66.0 C. The variation in melting temperatures was accomplished by replacing A and T bases (with two hydrogen bonds) with G and C bases (three hydrogen bonds) that would have higher thermal stability.
The results showed that by approaching the melting point of the duplex siRNA, a dramatic peak broadening occured that indicated on-column duplex melting to its corresponding two single strands. As the temperature was increased and exceeded that of the duplex melting point, the duplex quickly melted and the two complementary single stranded siRNA eluted as sharp peaks.
The authors also point out that the retention of siRNA sequences was affected by the sequence composition, whereas the duplex eluted in similar retention times.
Based on the duplex melting experiments, the authors selected 20 C as the standard separation temperature. The IP-RPLC results showed good separation of duplex from single-stranded impurities and no degradation of duplex was observed.
The results also showed that the IP-RPLC method was able to resolve siRNA from its truncated duplex impurities. These impurities are formed when annealing single stranded siRNA are contaminated with shorter failed sequences. Purification of siRNA in duplex form is a good option only if there is an efficient separation of truncated impurities from the target duplex.
In order to investigate the ability of the IP-RPLC method to effectively separate the truncated impurities from the target duplex, they prepared 5' truncated samples from one of the complementary strands by its partial enzymatic digestion. The mixture of the truncated impurities was then annealed with a full-length complementary strand. The resulting mixture resembles a crude synthetic mixture that is usually contaminated with truncated sequences.
The data showed that the IP-RPLC method separated the truncated duplexes. These were confirmed by mass spectrometry.
Upon closer inspection of the MS data, the authors noticed that the duplex mass was not observed, but rather the two complementary single strand masses were observed by MS. This indicated that the siRNA duplex is stable under the chromatographic separation, but duplex melts in the ESI source in the MS.
The authors further investigated the use of MS detection for intact duplex siRNA by decreasing both the desolvation and ESI source temperatures. The data showed some presence of duplex but mostly as hexylamine adducts. The use of ammonium acetate yielded better MS results for the analysis of duplex, however separation changed where duplex was now eluting before its corresponding single-stranded species and the separation efficiency decreased.
Next, the authors investigated the use of IP-RPLC method for the duplex siRNA purification. First, they prepared the duplex siRNA by annealing crude complementary single strands. The purification of the resulting duplex was successful and provided duplex sample with >98% purity.
After the successful purification of the duplex that was formed by annealing in a separate step, the authors studied the on-column formation of duplex where the two complementary strands were injected on the column at conditions below the duplex melting temperature. The results showed that the duplex is formed spontaneously and effectively.
The authors then scaled up the siRNA purification using the on-column annealing approach. The expected single and double stranded impurities eluted before the target duplex peak. The collected purification fractions were analyzed by UPLC method. The data confirmed the purity of the purified duplex.
Finally, the authors investigated the limitations of the IP-RPLC purification method. They found that oligonucleotides with strong secondary structures yield very broad peaks making it difficult to purify them using this method. Further investigation showed that the purification process was more successful if the secondary structure duplex was formed off-column.
The authors conclude that the methods they provide in this article can be used for preparative purification of siRNA in both single stranded and duplex forms. The proposed analysis and purification methods are fast, efficient and cost-effective.
Assignment #3: FAQ
Question: What is EndNote and how do I get this at Drexel?
Answer: EndNote is a commercial reference management software package by Thomson Reuters. It can be used to search for literature, develop and manage a personal library of references, and create and format citations when writing articles for publication. The software is available for download to all Drexel students.
How to download the software?
-Log in using your Drexel username and password
-Click on 'students' link
-Download the EndNote software (PC software or Macintosh software)
Here are the main features of EndNote software:
Ability to search bibliographic databases on the Internet
More than 3,900 files can be found.
EndNote's 'Search' function allows to search for different Internet databases, including Web of Science, PubMed, and the Library of Congress.
The references of interest can be directly exported from the Internet databases.
Organize references, PDFs, and any other files in a custom library
An unlimited number of libraries and of any size can be created.
Subsets or custom group references can be created for better maintenance and easier organization.
Full text articles can be located and downloaded automatically.
PDFs and other files can also be stored within the EndNote reference library.
The settings on the EndNote library display, such as bibliographic preferences and other options, can be easily organized from the 'Preferences' link.
EndNote includes more than 4,500 predefined bibliographical styles. Each style can be modified or new styles can be created.
Create instant bibliographies in Microsoft Word
As citations are inserted in your manuscript, a bibliography is automatically created.
References in the bibliography section are automatically updated as citation changes in the word document are made.
References can also be directly transferred from colleagues' papers in your EndNote library with the export traveling library feature.
For more information on EndNote software and tutorials, see
help on how to format text
Turn off "Getting Started"