Common Molecular Biology Techniques and Their Uses in Chemistry

Becky Farmer Long Literature Seminar August 25, 2008 What is Molecular Biology?

•Molecular biology is commonly defined as the branch of biology that examines biological phenomena at the molecular level

•Mainly concerned with the study of biological macromolecules, such as DNA, RNA and proteins and their various interactions

•Term “molecular biology” was coined in 1938 by Warren Weaver, the former director of the Division of Natural Sciences at the Rockefeller Foundation

•Characterized by several common techniques: •Polymerase chain reaction (PCR) •Polyacrylamide gel electophoresis and blotting •Fluorescence activated cell sorting (FACS) •Knockout mice •RNA interference (RNAi) •Microarrays •Confocal microscopy •Fluorescent in situ hybridization (FISH) •Expression cloning/cell transfection •And many others….. The Basics: DNA, RNA and Everything In Between

•The process by which genetic information is shared and expressed in the cell is known as the “central dogma of molecular biology”

•DNA  RNA  protein

•DNA is duplicated through the process of replication

•mRNA is made through transcription

•The genetic code is read from the mRNA by ribosomes, which synthesize proteins through the process of translation The Basics: DNA Replication

•Genetic information is passed on to new cells via the process of semi-conservative replication

•Each DNA strand containing the genetic code is copied by DNA polymerase III in a 5’3’ fashion .The leading strand is copied continuously, while the lagging strand is copied in short pieces (Okazaki fragments) and then ligated together

•DNA helicase unwinds the leading edge of the replication fork, while topoisomerase prevents tangling of the chromosomal material Polymerase Chain Reaction (PCR)

•Developed in 1983 by Kary Mullis, who received a Ph.D. in biochemistry from the University of California at Berkeley

•Mullis conceived of the idea for PCR while working for Cetus Corporation, a biotechnology company located in Emeryville, CA

•Mullis received the Nobel Prize in Chemistry for his work in 1993, the first prize ever awarded for research performed at a biotechnology company

•In the press release for the Nobel Prize, the Royal Swedish Academy of Sciences described the impact of PCR as follows:

Kary Mullis The biomedical applications of the PCR method are already legion. Now that it is possible to discover very small amounts of foreign DNA in an organism, viral and bacterial infections can be diagnosed without the time-consuming culture of microorganisms from patient samples. PCR is now being used, for example, to discover HIV infections. The method can also be exploited to localize the genetic alterations underlying hereditary diseases. Thus PCR, like site-directed mutagenesis, has a great potential within gene therapy. […] In police investigations PCR can give decisive information since it is now possible to analyze the DNA in a single drop of blood or in a hair found at the scene of a crime. Mechanism of PCR

•PCR is used to exponentially amplify a sequence of DNA, known as the target sequence

•Isolated DNA is added to reaction along with primers (complementary to flanking regions of target sequence), dNTPs, and heat-stable DNA Taq polymerase

•First step involves heating the reaction to ~95 °C for 20-30 seconds to denature the double helix

•Rapid cooling to 50-65 °C allows for annealing of primers

•Reaction is then heated to 72 °C, the optimal temperate for the Taq polymerase, which then synthesizes new DNA strands from primers in 5’3’ direction Mechanism of PCR Continued

•Another cycle then proceeds with the same temperature changes, or “steps”, with the newly synthesized DNA strands acting as templates

•After the second cycle, short strands containing only the DNA sequence of interest are formed

•These short strands can act as templates for further cycles, thereby leading to exponential amplification of the sequence of interest

•Consists of generally 20-40 cycles, with each cycle consisting of discrete temperature steps

•Ideal situation is amplification of 2n with n equal to the number of cycles .Amplification is a millionfold after 20 cycles and a billionfold after 30 cycles Quantitative Real-Time PCR (qRT-PCR)

•Quantitation of gene expression levels is important for understanding of cell biology

•Use of fluorescence emission generated during each cycle allows accurate quantitation of gene of interest

•Most commonly used method involves fluorescent probe that hybridizes to sequence of interest

•When Taq polymerase reaches probe, 5’ 3’ exonuclease activity destroys the probe and releases fluorescent dye

Mocellin, S.; Rossi, C. R.; Pilati, P.; Nitti, D.; Marincola, F. M. Trends Mol. Med. 2003, 9, 189-195. The Basics: Transcription

•Transcription is the process by which the genetic code in DNA is passed on to single- stranded mRNA .Particularly important step in gene expression since this is the level at which the cell regulates which proteins are made

•RNA polymerase (I, II or III) binds to a promoter in the DNA sequence with the assistance of multiple transcription factors and synthesizes the gene .Promoter usually 25 nucleotides upstream of the gene of interest

•DNA helix reassembles after transcription, while mRNA is extruded from the nucleus, post-transcriptionally modified and translated into protein Reverse Transcription PCR (RT-PCR)

•Reverse transcription is the process used by viruses to synthesize DNA from RNA genome

•Similar principle is used for reverse transcription PCR to convert RNA to cDNA

•Technique used for measurement of gene expression in the cell

•Often combined with quantitative real-time PCR for accurate picture of actions occurring inside cell

Bustin, S. A.; Mueller, R. Clin. Sci. 2005, 109, 365-379. Electrochemical Real-Time PCR

•Current real-time PCR protocols rely on bulky optical systems for detection of amplification

•For point-of-care applications, need an inexpensive portable device

•In the first step, PCR is carried out in solution phase to give dsDNA containing modified Fc markers

•The DNA is then annealed to an electrode containing complementary probes

•Extension occurs, increasing number of Fc markers on surface  electrical signal

Yeung, S. S. W.; Lee, T. M. H.; Hsing, I. J. Am. Chem. Soc. 2006, 128, 13374-13375. Electrochemical Real-Time PCR Continued

•Voltammetric scans taken in presence (sample) and absence (negative control) of target sequence demonstrate the buildup of Fc markers on glass electrode

•Current measured as a function of cycle number shows that signal is detected on a real-time basis

•Signal is detected more quickly than in traditional optical systems  could be used for detection of small amounts of DNA

Yeung, S. S. W.; Lee, T. M. H.; Hsing, I. J. Am. Chem. Soc. 2006, 128, 13374-13375. Polyacrylamide Gel Electrophoresis (PAGE)

•Gel electrophoresis is used to separate DNA, RNA or proteins by size and charge

•The gel is made of a crosslinked polyacrylamide, although it can also be made of agarose

•Current is applied to the gel, which causes the proteins/nucleic acids of interest to migrate toward anode/cathode  causes discrete bands which can be identified by size

•SDS-PAGE is used to analyze proteins, since sodium dodecyl sulfate denatures proteins Blotting

•Gel electrophoresis is the first step in the biological procedure known as blotting

•Once macromolecules of interest have been separated using gel electrophoresis, they are transferred to a separate surface where they are further analyzed

Edwin Southern

•Types of blots: .Southern blot – used to probe for a specific sequence of DNA, named after Edwin Southern, a British biologist who developed the technique in 1975

.Western blot – analysis of proteins via antibody detection

.Northern blot – detection of specific sequences of RNA The Basics: Translation

•Translation is the process by which the genetic code found in mRNA is used to synthesize proteins

•Codons (sequence of three nucleotides) are matched to their corresponding tRNA carrying the appropriate amino acid

•The ribosome catalyzes the elongation of the growing peptide chain and then transports the nascent protein into the endoplasmic reticulum, where it is folded into its 3D structure and post-translationally modified Western Blots

•Western blots are the most commonly used blotting technique in molecular biology

•Used to gauge the level of protein expression in the cell  since protein levels are a result of gene expression, Western blot gives accurate depiction of gene activity in the cell

•First developed by George Stark at Stanford University, then later perfected and termed “western blot” by W. Neal Burnette in 1981

•Western blotting is used to diagnose several diseases, including HIV and bovine spongiform encephalopathy Western Blots in Hsp90 Inhibitor Evaluation

•Hsp90 is one of the most abundant proteins in humans  accounts for 1-2% of all protein

•Assists in protein folding as a chaperone and is also important in tumor progression .Stabilizes mutant oncogenic proteins such as p53 and bcr/abl .Maintains PI3K and AKT, both of which inhibit apoptosis .Promotes metastasis (MMP-2) and angiogenesis (NOS and VEGF)

Yu, X. M.; Shen, G.; Neckers, L.; Blake, H.; Holzbeierlein, J.; Cronk, B.; Blagg, B. S. J. J. Am. Chem. Soc. 2005, 127, 12778-12779. Hsp90 Inhibitor Development Continued

•Each compound was tested for its ability to cause degradation of Hsp90 client protein phospho-AKT in SKBR3 breast cancer cells

•Geldanamycin was used as positive control

•SAR shows several patterns: .7-position of coumarin ring is important for activity (B vs. D/E) .Compounds with amide linker show more potent action (A) .Diol may mimic ATP in binding site (4)

•Compound A4 was identified as the most potent inhibitor

Yu, X. M.; Shen, G.; Neckers, L.; Blake, H.; Holzbeierlein, J.; Cronk, B.; Blagg, B. S. J. J. Am. Chem. Soc. 2005, 127, 12778-12779. Hsp90 Inhibitor Development Continued

•Novobiocin analog A4 also tested to determine their action against hormone receptors

•Two cell lines were used: LNCap (mutated androgen receptor PCa) and LAPC-4 (wild-type androgen receptor PCa)

•A4 causes degradation of androgen receptor (AR), AKT and HIF-1α at low concentrations (~1µM)

Yu, X. M.; Shen, G.; Neckers, L.; Blake, H.; Holzbeierlein, J.; Cronk, B.; Blagg, B. S. J. J. Am. Chem. Soc. 2005, 127, 12778-12779. Fluorescence Activated Cell Sorting (FACS)

•Fluorescence activated cell sorting, or FACS, is a technique used to rapidly separate cells based on a specific physical property

•Fluorescent tags are used to identify specific markers on cells (often cell surface markers)

•Developed by Leonard Herzenberg at Stanford University in 1969 .Used the technology to separate hamster ovary fibroblasts from a mixture of mouse splenocytes .Later in 1972 separated cells labeled with fluorescent antibodies

•Often called flow cytometry, but is really a subset of the wider field of flow cytometry

Delude, R. L. Crit. Care Med. 2005, 33, S426-S428. Mechanism of FACS

•A population of cells is tagged with fluorescent antibodies against a particular cellular marker of interest

•The cells then pass into a narrow stream of liquid and vibration causes the stream to break into droplets (one cell per droplet)

•The fluorescence emission of each cell is measured, then a charge is applied to the droplet based on the fluorescence signal

•An electric field is applied and cells are sorted based on charge/fluorescent signal The Basics: Apoptosis

•Apoptosis is a form of “programmed cell death” by which a cell can commit suicide upon incurring significant damage or when it is no longer needed

•Normal process that takes place during human development, but often is subject to dysregulation in cancer cells

•Regulated by the caspases, a family of aspartate-specific cysteine proteases specifically used for this purpose

•Characterized by DNA cleavage, cell blebbing, nuclear degradation and eventual formation of apoptotic bodies

Ashkenazi, A.; Herbst, R. S. J. Clin. Invest. 2008, 118, 1979-1990. FACS in Chemistry: Nano-Flares

•Developed oligonucleotide functionalized gold nanoparticles that act as probes for intracellular RNA

•Gold particle quenches fluorescent “flare” until probe hybridizes to target RNA sequence

•Probes were designed to be complementary to survivin and were tested in SKBR3 cells

•Fluorescence signal is 2.5x higher in cells labeled with survivin particles than with noncomplementary probe sequence

Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. J. Am. Chem. Soc. 2007, 129, 15477-15479. Knockout Mice

•Knockout mice (or transgenic mice) are animals that have been genetically engineered to carry a modified or deleted gene

•The 2007 Nobel Prize in Physiology or Medicine was awarded to Drs. Mario Capecchi (University of Utah), Martin Evans (Cardiff University) and Oliver Smithies (UNC) for their pioneering work to develop knockout mice

•First report of generating gene-targeted mice was in 1989  since then, more than half of the mammalian genome (over 10,000 genes) have been knocked out

Mario Capecchi Martin Evans Oliver Smithies Generation of KO Mice

•Embryonic stem cells are cultured from mouse blastocysts and are electroporated with the modified gene of interest

•Engineered genes insert into the host cell genome via the process of homologous recombination

•Usually the engineered gene contains a resistance element which allows for selection of cells which are successfully transfected  most commonly a neomycin resistance element

•Cells which have been transfected are then isolated by positive-negative selection strategy Positive-Negative Selection Process

•The targeting vector contains both the neomycin resistance element and a HSV thymidine kinase domain

•When the targeting vector inserts into the genome via homologous recombination (pathway A), only the neomycin resistance element will be incorporated

•If random integration occurs (pathway B), then both elements will be incorporated

•Transfected cells can then be selected by their resistance to neomycin (positive) and their absence of thymidine kinase activity (negative) Generation of KO Mice Continued

•Once clones of transfected cells have been cultured and isolated, they are then injected into mouse blastocysts

•The modified blastocysts are implanted into female mice, which give birth to chimeras

•These chimeras are then mated with other mice, giving both normal mice and mice carrying a full copy of the targeted gene

•Heterozygous mice are then inbred with one another to yield homozygous mice in a Mendelian fashion Using KO Mice to Test MRI Contrast Agents

•Most gadolinium-based contrast agents are too large to fit in the microvasculature of an atherosclerotic plaque

•This group developed an HDL-like nanoparticle contrast agent

•Advantages to traditional imaging: .HDL-like particles naturally localize to atherosclerotic plaques .Particles with endogenous components do not trigger immune reactions .Small size (7-12 nm) .High contrast agent load

•HDL apoproteins combined with lipid-based contrast agent Gd-DTPA-DMPE and fluorescence marker NBD-DPPE

Frias, J. C.; Williams, K. J.; Fisher, E. A.; Fayad, Z. A. J. Am. Chem. Soc. 2004, 126, 16316-16317. Contrast Agent Imaging Studies

•Hyperlipidemic mice (apoE-knockouts) were used as a model for atherosclerosis

•Contrast agent was shown to localize to the atherosclerotic plaques approximately 24 hours after injection and disappeared at 48 hours

•Aortas were removed at 24 hours and imaged by confocal fluorescene microscopy  HDL contrast agents localized to intimal layer inside macrophages

Frias, J. C.; Williams, K. J.; Fisher, E. A.; Fayad, Z. A. J. Am. Chem. Soc. 2004, 126, 16316-16317. RNA Interference (RNAi)

•RNA interference was developed by Craig Mello (University of Massachusetts) and Andrew Fire (Stanford University)

•Awarded the 2006 Nobel Prize in Physiology or Medicine for their work on RNAi  only 8 years after publication!

•Showed that injection of dsRNA against mex-3 gene produced almost complete elimination of gene expression in C. elegans embryos (panel d) .Antisense injection (panel c) did not show as complete gene “knockdown” Craig Mello and Andrew Fire

Fire, A.; Xu, S.; Montgomery, M. D.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Nature 1998, 391, 806- 811. Mechanism of RNAi

•Long dsRNA is introduced into the cell and is recognized as foreign by the Dicer enzyme

•dsRNA cleaved into siRNA (small- interfering RNA) which associates with RISC (RNA-induced silencing complex)

•RISC unwinds siRNA and presents the sequence in the cytoplasm

•Complementary mRNA found in the cell binds siRNA bound to RISC  mRNA degraded by Argonaute enzyme

•This mRNA degradation leads to gene silencing seen with RNAi RNAi in Chemistry: Nano-Flares Continued

•siRNA experiments were performed against survivin to determine if nano-flares could react to changes in transcript level

•Flow cytometry shows that siRNA treated cells have smaller fraction of cell population exhibiting fluorescence

•Decrease in fluorescence is in agreement with transcript levels as determined by RT- PCR

Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.; Mirkin, C. A. J. Am. Chem. Soc. 2007, 129, 15477-15479. Gene Microarrays

•Gene microarrays are a high-throughput version of the Southern blot  used to identify specific sequences of DNA in a parallel fashion

•Microarray technology is common used to profile gene expression or to identify single nucleotide polymorphisms (SNPs) within the genome

•The first report of using miniaturized microarrays for gene expression profiling was reported in Science in 1995

•An entire eukaryotic genome (!) for Saccharomyces cerevisiae (yeast) was published in 1997

Lønning, P. E.; Sørlie, T.; Børresen-Dale, A. L. Nat. Clin. Pract. Oncol. 2005, 2, 26-33. Performing a Microarray Experiment

•Most gene array experiments are two- color detection experiments  each sample is identified by a different color fluorescent probe

•Two cell samples are prepared and the RNA is isolated from each

•RNA is converted to cDNA via reverse transcription with fluorescently tagged nucleotides

•cDNA is hybridized to microarray chip, which is read by fluorescence detection

Lønning, P. E.; Sørlie, T.; Børresen-Dale, A. L. Nat. Clin. Pract. Oncol. 2005, 2, 26-33. Using Microarrays to Determine % ee

•Researchers named new technique “reaction microarrays” and modeled system after DNA microarray technology

•System used to measure ee of thousands of samples simultaneously

•Amino acid samples hybridized to amine-functionalized slide, then treated with pseudoenantiomeric fluorescent probes 1 and 2

•Ratio of red to green fluorescence determines % ee

Korbel, G. A.; Lalic, G.; Shair, M. D. J. Am. Chem. Soc. 2001, 123, 361-362. Using Microarrays to Determine % ee

•Fluorescence could be used to detect ee within ±10% ee in 92% of samples

•High-throughput analysis on microarray allowed identification of >99% ee samples of proline out of 15,552 samples in 48 hours

Korbel, G. A.; Lalic, G.; Shair, M. D. J. Am. Chem. Soc. 2001, 123, 361-362. Determination of Protein-Ligand Interactions

•To address need for screening of multiple compounds simultaneously, the Schreiber group developed small molecule printing (SMP)

O •Compounds are attached to maleimide- O OH rich glass slides via a conjugate addition O Me

NH  thioether linkage 1a,b Me H HN H 2a,b O H H OH R R S N •Molecules were chosen based on their H H H O O known interaction with specific proteins: Me OMe Me N .Biotin (1a,b)  Streptavidin 4a OMe .Digoxigenin (2a,b)  DI-22 O R Me O 3a,b (antibody) N O O Me N .Pipecolyl α-ketoamide (3a,b)  H R CO2 O O O FKBP12 (immunophilin) Me O Me Me X O H N a X=SH N CO2H H b X=H

MacBeath, G.; Koehler, A. N.; Schreiber, S. L. J. Am. Chem. Soc. 1999, 121, 7967-7968. Determination of Protein-Ligand Interactions

•Each compound was spotted onto a maleimide- derived slide using specific spatial locations

•Fluorescently labeled proteins were then added to the plates and system was scanned for fluorescence

•Spots localized to specific molecules of interest only when thiol functionality was present

•Molecules were attached to an array containing 10,800 individual spots  each compound was successfully identified in a single step

MacBeath, G.; Koehler, A. N.; Schreiber, S. L. J. Am. Chem. Soc. 1999, 121, 7967-7968. Conclusions

•Molecular biology is a diverse and complex scientific discipline  extremely technique-driven

•Several techniques have come to the forefront as the most commonly used and most powerful: .RNAi .Knockout mice .PCR .FACS .Microarrays .Blotting

•Important molecular biology techniques can be used to influence chemistry and vice versa

•An understanding of biological techniques is an essential part of the chemist’s toolbox, since molecular biology is used extensively in pharmaceutical testing and compounds are designed to target biological systems