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21 Pages
Presentation, Melissa Medley
Course: WS 5320, Fall 2008School: Texas Tech
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Word Count: 633
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TINY RNAI: PIECES LEAD TO BIG CHANGES A Melissa Medley Presentation for Dr. Lou Densmores Molecular Biology Class The Basics RNAi-RNA interference siRNAs-small interfering RNAs miRNAs-micro RNAs Both involve binding of small, single stranded RNA molecules to mRNAs as a means of controlling translation Cellular Applications Regulation Suppression Activation Development Disease Treatment (will discuss later)...
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TINY RNAI: PIECES LEAD TO BIG CHANGES
A Melissa Medley Presentation for Dr. Lou Densmores Molecular Biology Class
The Basics
RNAi-RNA interference
siRNAs-small interfering RNAs miRNAs-micro RNAs
Both involve binding of small, single stranded RNA molecules to mRNAs as a means of controlling translation
Cellular Applications
Regulation
Suppression Activation
Development Disease Treatment (will discuss later)
miRNAs
Come from longer pre-micro RNAs Cleaved by Drosha protein Dicer protein recognizes and cleaves further Complementarity not necessary
http://www.microrna.ic.cz/obr/image003.png
siRNA
Starts as dsRNA Made into RISC (RNA Induced Silencing Complex) siRNA anti-sense binds to mRNA Ago 2 endonuclease activity cleaves dsRNA, has slicer and PAZ domains too
http://escience.ws/b572/L26/images/sirna_prinzip.gif
http://www.medgadget.com/archives/img/sirna.jpg
RISC Load Complex (RLC)
Loads siRNA onto Dcr-1 Helps to determine which strand is loaded R2D2 helps unwind dsRNA R2D2 associates with Dicer and cleaves siRNA Ago2 replaces R2D2/Dcr-1 heterodimer
Dicer
Important in processing of miRNAs and siRNAs Important in RISC complex activity Has helicase and RNase activities Linked to important brain development activities
http://img.alibaba.com/photo/51013285/Nicer_Dicer_with_VCD_HY_4902_.jpg http://www.hhmi.org/news/popups/images/doudna01132006_380.gif
Therapeutic Uses
Transposon defense RNA viruses as well as others Parasitic Defense Parkinson's and other neurological disorders Cancer ??????
siRNAs Classes
Three classes Determined by 5 and 3 end sequences Class I has two sub classes, Ia and Ib Class III is opposite from Class I Class II is what doesnt fit in Classes I or III
Other Factors
Protein binding Cellular location mRNA secondary structure Length of mRNA sequence
G=-RTlnK
K1=G duplex K2=G target structure K3=G intra-oligomer K4=G inter-oligomer K0= G siRNA duplex=K1
Equilibrium considered in the OligoWalk algorithm for predicting the affinity of a structured oligonucleotide (siRNA) to a structured target (mRNA)
Lu, Z. J. et al. Nucl. Acids Res. 2008 36:640-647; doi:10.1093/nar/gkm920
Copyright restrictions may apply.
siRNAs Vectors
Chemical Product In vitro transcription enzymatic and degradation Endogenous expression
Plasmid vectors Viral Vectors Inducible expression vectors
http://www.medgadget.com/archives/img/654324sirna.jpg
Problems with Therapy
Mutation How do we get it inside?
lipids
siRNA are not as specific as some think
Unintended Silencing Unintended Activation
Sources
Li, John Ahi; and David H. Mathews. Efficient siRNA Selection Using Hybridization Thermodynamics. Nucleic Acids Research. Vol 36, No. 2, pages 640-647: December 10, 2007. DePaula, Daniel; M. Vitria L.B. Bentley, and Ram I. Mahato. Hydrophobization and Bioconjugation for Enhanced siRNA Delivery and Targeting. RNA, Vol 13, Pages 431-456; February 28, 2007. Tomari, Yukihide; Christian Matranga, Benjamin Haley, Natalia Martinez, and Phillip D. Zamore. A Protein Sensor for siRNA Asymmetry. Science Vol 306, no. 5700, pages 1377-1380; November 2004. Ui-Tei, Kumiko; Yuki Naito, Fumitaka Takahashi, Takeshi Haraguchi, Hiroko Ohki-Hamazaki, Aya Juni, Ryu Ueda, and Kaoru Saigo. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acid Research Vol 32, No. 4, 2004. Jackson, Aimee L; and Peter S. Linsley. Noise amidst the silence: off target effects of siRNAs? Trends in Genetics. Vol 20, No. 11, November 2004. Cuellar, Trinna L.; Tigwa H. D...
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