Hi All =)
I will be in lab Saturday (Sept. 11) from 12-5 and Sunday (Sept. 12) 2-6.
Xinh
New 100bp Ladder Diluted and Functional
The new Invitrogen 100 base pair was tested during my R16 cycle course PCR.
The ladder can be seen here, along with third wash samples from six, nine, twelve, fifteen, and twenty cycles of PCR. As N58 RNA is slightly larger than 100 base pairs, its location just above the lowest ladder band is an indication of its effectiveness. Multiple 20 uL aliquots were created and placed in the -20 freezer. The stock ladder solution, previously diluted by Brad, was placed in the ladders and primers freezer box, distinguished by red tape.
Just getting Started
I was reading a pretty interesting post over at Lifehacker about getting started and how it is overrated. I would have to agree/disagree. I think for you all, just getting into lab and starting with your selections is the first step and should not be overlooked. Just start doing it and you'll get back into it easy. But, the point of the Lifehacker disagreement was more about long-term success. These two points are suggested:
- Establish, over time, a deep emotional conviction that you want to follow the pursuit.
- Build an exhaustive understanding of the relevant world, know why some succeed and others don't, and know exactly what actions are required.
- Just start on whatever target you want till you feel comfortable doing it. Or start on the homework by reading the questions, then read the chapter.
- Be proud of your selection and target, own it. Or be proud of the work you turn in for a grade. If you have ownership, you will feel more responsible for the outcome and thus work diligently for the results.
- Know why other succeed at a topic, selection, etc and why some have failed. Do the things that work for you.
- Understand the literature (goes beyond journal club). Learn Learn Learn. You will never stop learning, so use college to find out the best method that motivates your learning.
Nucleic Acid Aptamer Selection against TfR for the Therapeutic Treatment of Lysosomal Storage Diseases
Lysosomal Storage Diseases are inherited metabolic disorders that result from dysfunctional lysosomal enzymes. These enzymes facilitate the digestion of macromolecules within eukaryotic cells (Fong 2010). The absence of functional lysosomal enzymes results in the accumulation of macromolecules within the cell and can lead to many harmful effects on cells, tissues and organs (Futerman 2004). An efficient method of enzyme delivery could alleviate many of the problems associated with these diseases. The selection of an aptamer against a target membrane bound protein involved in endocytosis could provide efficient system of enzyme delivery to lysosomes. Post-selection modification of the aptamer would be necessary to link an enzyme to the aptamer. This method of aptamer-mediated endocytosis and post-selection modification could have a significant impact on the future of targeted enzyme delivery, as the design is very versatile.
Figure 1. Transferrin receptor-mediated endocytosis of Fe3+.
The transferrin receptor (TfR) is a membrane bound protein that facilitates the transport of iron into cells (Ray 2010). The binding of transferrin-Fe+3 to TfR signals for endocytosis of the entire molecule [1]. The molecule is transported to the endosomal compartment, iron is released and the rest of the molecule is incorporated back into the membrane. An aptamer will be selected against the transferrin receptor and modified post-selection with the addition of a functional lysosomal enzyme. This will enable the delivery of functional enzymes to lysosomes through aptamer-mediated endocytosis.
Though this form of enzyme replacement therapy is relatively new, there have been successful results, indicating that this therapeutic application of aptamers may be key to the future of lysosomal enzyme delivery. Researchers at UCLA have demonstrated the delivery of enzymes to cells deficient in lysosomal enzymes using the aptamer-mediated endocytosis method (Neufeld 2007). Chen et al. selected an aptamer against TfR and modified the aptamer with the attachment of α-l-iduronidase, a lysosomal enzyme. This aptamer complex was taken up by α-l-iduronidase-deficient mouse fibroblasts. The selection of an aptamer against TfR for aptamer-mediated endocytosis could have a revolutionary impact on the delivery of enzymes and treatment of various lysosomal storage disorders.
Human TfR from ARP- $259/ 50ug- http://www.biocompare.com/ProductDetails/1537096/Human-Transferrin-Receptor.html?
Chen, Chi-hong B. et. al. "Aptamer-based Endocytosis of a Lysosomal Enzyme." PNAS 105.41 (2008): 15908-5913.
Fong, Chin-To. "Lysosomal Storage Disorders." Merck & Co., Inc. Feb. 2010. Web. 04 Sept. 2010.
Futerman, Anthony H., and Gerrit Van Meer. "The Cell Biology of Lysosomal Storage Disorders." Nature 5 (2004): 554-65.
Neufeld, Elizabeth F. "Aptamer Mediated Correction of Lysosomal Enzyme Deficiency." The Regents of the University of California. UCLA,
Ray, Partha, and Rebekah R. White. "Aptamers for Targeted Drug Delivery." Pharmaceuticals 3 (2010): 1761-778.
Image from: http://flipper.diff.org/app/pathways/info/1562
Files uploaded on blackboard
I have posted the first two lectures under "Course Documents" within blackboard. If you also didn't know I posted the Syllabus last week under "Syllabus".
Targets Ordered
The following targets have been ordered:
- HIV-gag p24 (ordered last Monday) - Arrived 9-8-2010!!!
- Dengue Virus Envelope (ordered last Monday)
- S100B
- INF-g
- CXCL1
- S100A4
- APP - B-amyloid precursor protein
The following targets are already in lab
- a-synuclein
- EphA2
- b-lactamase
- transferrin
- BPS 1027 and HCP1
- Fluorescent proteins
I did not order MUC-1 or SATB1 because I have to talk with Sabeena and Lola (respectively) - or look at the comments on your abstracts and respond...
Potential Targets
Due to collaborations, I can't provide too much information here on the blog, but would be happy to discuss with anyone interested in the following targets to please see me.
FGF-8b
IL21R
Bcl-2
MMP-7
To functionalize a target
We typically use a biotinylated NHS ester that will bind to amines (R-N-H2 groups). Pierce makes the EZ-Link NHS-SS-Biotin. For this to work, you must have a target with an N-terminal amine (i.e every polypeptide) or a protein with an amino acid that contains amines (i.e. cysteine). The problem with functionalizing proteins this way is that you have to have the ratio of reagent to target just right to ensure you are attaching one or two biotins to the protein without wasting to much non-functionalized protein or biotinlyating the crap out of it. In addition, it can be difficult to assay the concentration properly.
Binding Assay 1 and 2
Binding Assay Data 1 Performed 6.21.2010
The first binding assay showed progressive binding through the rounds with a decrease at round 5 and an increase at round 7B. When restarting selection after a brief 2 week hiatus, it was discovered that the DNA in round 5 (1) had degraded into a short unusable strands and therefore, selection had to be restarted at round 3 and then progressed with parallel selections (A and B) per selection, per round. There is not a great amount of deviation from the average, which shows promise that the percentage binding is accurate. The green bar indicates the percentage of total binding species while the red bar indicates the total percentage of species that bind to things other than the protein.
Binding Assay 2 Performed 7.10.2010
This binding assay shows that with progressive rounds after round 7, there has been an increase in background binding, relative to the total binding. The assay for round 10 was not completed without problems due to an odd smear on the cartridge, despite multiple tries with different Storm Imagers and different cassettes. The cassettes and the screen were exposed for approximately 30 minutes each due to the high radioactivity of the P-32 used during the assay. Round 7 shows the most promise for the A round and will later be sequenced. When visualizing the cassette, there was such overexposure of the bottom where the nonbinders had washed out that it had essentially bleached the top part white. The pixel reader, however, can determine the differences of minute shades of pixels, so percentage binding can still be achieved. The original assay was supposed to be performed with rounds 7A, 8B, 8A, 10B, 10A, but due to the low yield after the etOH precipitation, it was impossible to continue. The top half of the cassette shows the binders for the assay with 1027 protein. The bottom half of the cassette shows that which was done without the 1027 protein. The blots in the top grid (1) cannot be seen, but based on the bottom grid (2). Grid 2 represents the washed away non binders. The darker blots on the bottom half of the grid show that more has washed out during the negative protein assay, which is promising. The green bars indicate percentage of total binding species and the red bars indicate percentage of total nonspecific binding species.
Lab this week?
Just wondering if we could work with target's, other than the one we had last semester, to get back in the swing of things. I was in lab today, but couldn't find any fibrinogen in the freezer (assuming we are out); can I practice on a different target (HBS and Angiotensin are still available) before I start work with my proposed one?
Figures in your proposals
Make sure to make a connection from the figures in your proposals to what you are saying. There should not be anything that "takes up space" it should have a purpose stated with your writing.
Therapeutic Development of an Aptamer against the Dangerous side of SATB1
Proposal Placed in Dropbox. The link is http://dl.dropbox.com/u/11745574/Proposal---Aptamer%20Stream%20%28September%202010%29%207.docx
Figure 1:http://en.wikipedia.org/wiki/File:Protein_SATB1_PDB_1yse.pngIn vitro studies that have been conducted showed a reduction in SATB1 expression in highly metastatic cell lines reduced the invasiveness of those cells as well as their capability for unattached growth. Aggressive cancer cells need unattached growth in order for them to move through the blood and lymph vessels. (2) This in fact proves that the inhabiting of SATB1 will hinder it from increasing the growth of aggressive cancer tumors. There are no known successful aptamers that have been developed as therapeutics against SATB1.
An aptamer if successful would be very useful as a therapeutic against the SATB1 protein. Inhibiting the functional pathway of SATB1 would prevent its reprogramming of the genome to change the expression of genes that encourage the metastasizing of breast cancer cells. With that said, the aim is to develop an aptamer with a high enough affinity of specificity to bind to SATB1 in order to prevent it from being a “master regulator”. Like most anti-cancer drugs any therapeutic development against SATB1 expressed by breast cancer cells will also have an adverse effect on normal cells. Researching what types of cells are likely to express SATB1 could be useful in terms of a preventative measure. In vitro selection would be the method used to select against SATB1. Figure 2 is an outline of what is discussed in this abstract.
FIGURE 2) When Breast cancer cells express SATB1 protein, SATB1 reorganizes hundreds of genes thereby leading to the metastasizing of breast cancer cells. The inhibiting of SATB1 could prevent this from happening.
I came upon this on accident:
I was looking through CNN and this was in today's headlines, I though some of you might find it interesting. :)
Binding assay to determine binding affinity between N34 RNA and EphA2
Lack of binding between the ephrin A1 ligand and the EphA2 receptor, 130 kDa tyrosine kinase receptor found in adult human epithelial cells, causes unstoppable cell growth, and subsequently, development of tumors associated with epithelial cancers (Kinch, 2005; Ansuini et al, 2009). Inhibiting the kinase activity and phosphorylation of EphA2 protein receptor has been associated with a decrease in the growth of malignant cells (Ansuini et al, 2009). Ligand based approaches have been utilized by attaching a ligand to phosphorylating EphA2 protein which inhibits phosphorylation and subsequently decreases tumor growth (Walker-Daniels et al, 2002). A synthetic, highly modifiable ligand that can be used to inhibit EphA2 phosphorylation is an aptamer. Aptamers are nucleic acid fragments that have specific quaternary structures allowing them to bind with high specificity and affinity to certain protein targets, much like innate ligands, such as ephrin A1 (Phillips et al, 2008). Specific aptamers are filtered from a large nucleic acid pool through repeated, increasingly stringent tests, such as SELEX, which select for the highest affinity binders and amplification of those binders, and a binding assay can be performed to determine the progression of binding affinity through the rounds of selection.
A binding assay was performed to determine the affinity of filtered N34 RNA from R1, R3, and R5 to EphA2. 20 pmol of RNA, 40 pmol of EphA2 protein, and 200 pmol tRNA was used for the triplicate reactions which tested for both positive and negative binding. In order to detect the RNA, the double stranded DNA was labeled with radioactive beta 32P nucleotides during transcription. The radioactively labeled RNA was pipetted onto a nitrocellulose filter using the layout in figure 1. A nitrocellulose filter was used to filter the bound RNA and protein while a nylon filter collected the unbound RNA, as can be seen in figure 2.
Figure 1 Binding Assay Layout: Triplicate reactions were filtered using a nitrocellulose filter. To test for positive binding, R1, R3, and R5 protein and RNA was placed in three wells in the regions 1, 3, and 5 respectively. To determine how much of the binding was a result of RNA affinity for the nitrocellulose, R1, R3, and R5 RNA minus protein was placed in three wells in regions 2, 4, and 6 respectively.
Figure 2 Binding Assay: Nitrocellulose filter was used to separate the protein and bound RNA away from the unbound RNA which was collected on the nylon filter. The dots are a result from exposure of the 32P labeled RNA to phosphor plates.
Binding was minimal as shown by the faintness of the samples collected on the nitrocellulose filter in figure 2 and the comparison of the percent binding in figure 3. However, it must be noted that the results may be slightly skewed due to prolonged exposure between the 32P labeled RNA and phosphor plates which may account for the increased desensitization, and subsequently higher percent bound volumes, on the nitrocellulose filter. Nevertheless, the decreased average percentage of negatively binding RNA on the nitrocellulose filter (1.125% to 0.584%) does indicate the RNA with higher affinity for the protein, rather than the beads or filter, is gradually being selected. The average percent bound of high affinity RNA can be increased by progressively more stringent selection conditions to further reduce the amount of low affinity sequences and background binders.
Figure 3 Percent Binding: Percent binding is the amount of bound RNA and EphA2 on the nitrocellulose filter. It is calculated by dividing the total volume of protein and both bound and unbound RNA by the volume of bound RNA and EphA2.
Ansuini, H., et al (2009) “Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer.” Journal of Oncology 2009: 1-10.
Kinch, M. S. (2005). Targeted drug delivery using EphA2 or EphA4 binding moieties. Patent No. 20050153923. Laytonsville, MD, US.
Phillips, J. A., et al (2008) “Applications of aptamers in cancer cell biology.” Analytica Chimica Acta 621 Review: 101-108.
Walker-Daniels, J., et al (2003) “Differential Regulation of EphA2 in Normal and Malignant Cells.” American Journal of Pathology 162: 1037-1042.
Don't cite Wikipedia
Please do not cite Wikipedia. Wikipedia is not a credible source and some of the information on the pages is wrong. You may use Wikipedia to find additional references. Read those references and then cite that, if appropriate.
List of questions:
How does one functionalize a protein?
How do you know what beads to use? What is the significance to the different tags?
How do you know what beads to use? or columns? or anything else?
Does it matter what pool we use?
What is a good starting amount of pmol?
Do we need to start a new notebook, or can we use the old one?
Is the buffer they mention on the data sheet the one we should use?
*Reminder for Brad: Please send terrible binding assay results to Michael and I! :)
Volunteering for the Picnic
Please respond in the comments if you want to volunteer to work at the picnic. Information was posted here. So far I have Ashley and Katherine so far.
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