Download In Vitro

Chapter 5
RNA-Based technology
國立台灣海洋大學養殖系
呂明偉
1. Antisense RNA
2. RNAi
3. Ribozyme
FIGURE 5.1
Antisense RNA Is Complementary to Messenger RNA
Transcription from both strands of DNA creates two different RNA molecules, on the left, the
messenger RNA, and on the right, antisense RNA. These two have complementary sequences and
can form double-stranded RNA.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
2
RNase H recognize 7-base pair heteroduplex
Need not very long
Neurospora. frq gene.
FIGURE 5.2
Antisense RNA Blocks Protein Expression
(A) The complementary sequence of antisense RNA binds to specific regions on mRNA. This can
block the ribosome binding sites or splice junctions. (B) Antisense DNA targets mRNA for degradation.
When antisense DNA binds to mRNA, the heteroduplex of RNA and DNA triggers RNase H to
degrade the mRNA.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
3
FIGURE 5.3
Making Antisense in the Laboratory
(A) Antisense oligonucleotides. Small oligonucleotides are synthesized chemically and
injected into a cell to block mRNA translation. (B) Antisense genes. Genes are cloned in
inverted orientation so that the sense strand is transcribed. This yields antisense RNA that
anneals to the normal mRNA, preventing its expression.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
4
DNA is stable but needs modification
Nuclease, RNase H
Morpholino
PNAs
FIGURE 5.4
Modifications to Oligonucleotides
Replacing the nonbridging oxygen with sulfur (upper left) increases oligonucleotide resistance to
nuclease degradation. Adding an O-alkyl group to the 2’-OH on the ribose (upper right) makes the
oligonucleotide resistant to nuclease degradation and also to RNase H. Morpholino-antisense
oligonucleotides and peptide nucleic acids are two more substantial changes in the standard
oligonucleotide structure (lower left and lower right). Both are resistant to RNase H degradation. The
RNase H resistant oligonucleotides are used to target splice junctions or ribosome binding sites in
order to prevent translation of their target mRNA.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
5
FIGURE 5.5
Chimeric Oligonucleotides Use RNase H Sensitive Cores to Degrade Target mRNA
Chimeric oligonucleotides are made using different chemistries. The core region maintains RNase H
sensitivity, whereas the outer regions are RNase H resistant. When the oligonucleotide hybridizes to
target molecules in the cell, RNase H will digest the hybrid of oligonucleotide plus mRNA only where
the central domain forms a heteroduplex. RNase H will not digest any nonspecific complexes between
the chimeric oligonucleotide ends and the wrong mRNA.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
6
FIGURE 5.6
Antisense Oligonucleotides Correct Splicing Errors
(A) Beta-thalassemia is a blood disorder where extra splice sites are found between exon 2 and exon
3 (middle). Antisense oligonucleotides that block the extra splicing junctions restore the original
structure of the gene during splicing (right). (B) The Bcl-x gene makes two different proteins through
alternate splicing. Bcl-xS is made in a normal defective cell and promotes cell death via apoptosis
(left). Some cancerous cells do not produce Bcl-xS. Instead, the longer form, Bcl-xL, is produced via
alternate splicing, and it protects the cancerous cell from apoptosis (middle). To resensitize the
cancerous cell to apoptosis, antisense oligonucleotides that block the splicing junction for Bcl-xL
restore Bcl-xS protein production and, ultimately, sensitivity to apoptosis (right).
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
7
FIGURE 5.7
Oligonucleotides May Have Nonspecific Effects
(A) Misdirected RNase H cleavage can occur if the oligonucleotide recognizes a short seven basepair region of homology. (B) Chimeric oligonucleotides prevent nonspecific RNase H degradation.
When the RNase H resistant ends of the chimeric oligonucleotide bind to the wrong mRNA, the
structure of the oligonucleotide prevents RNase H from recognizing and cutting the heteroduplex.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
8
FIGURE 5.8
Methods of Antisense Oligonucleotide Uptake by Cells
(A) Liposomes are spherical structures made of lipids and cholesterol. Oligonucleotides are either
encapsulated in the central core or ride on the exterior surface of the liposome. The complexes enter
the cell via endocytosis and are released into the cytoplasm. (B) Basic peptides are naturally occurring
proteins that normally enter the nucleus of target cells. The oligonucleotide can be fused to these
peptides and ride into the nucleus with the basic peptide. (C) Streptolysin O, a toxin from Streptococci
bacteria, aggregates at the membrane to form a porelike structure. The oligonucleotides can pass into
the cell through the pore.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
9
FIGURE 5.9
Microinjection and Scrape-Loading
(A) Oligonucleotides can be injected directly into a cell using a very fine needle. Microinjection can be
done on individual cells grown in culture. (B) Scrape-loading is a mechanical method of getting
oligonucleotides into cultured cells. As the cells are scraped off the bottom of the culture dish, the
membranes break open, allowing the oligonucleotides to enter. When the cell membrane reseals, the
oligonucleotides are trapped within the cells.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
10
FIGURE 5.10
Cellular Mechanism of RNAi
(A) Double-stranded RNA triggers RNA interference. dsRNA is produced by RNA viruses during
infections, microRNA encoded by the genome, or overexpression of transgenes. (B) RNA interference
degrades all the RNA that is complementary to segments of double-stranded RNA. First, Dicer
recognizes dsRNA and cuts it into pieces of 21–23 nucleotides. A kinase phosphorylates the 5’ end of
each piece. Next, RISC unwinds the siRNAs and uses one strand to search out complementary
mRNA, which is degraded by associated enzymes.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
11
FIGURE 5.11
Amplification of RNAi
After RISC-associated enzymes cleave the complementary mRNA in a cell, another enzyme, RNAdependent RNA polymerase, binds to some of these fragments. RdRP synthesizes complementary
strands, making more double-stranded RNA. Dicer recognizes these fragments and creates more
siRNA.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
12
FIGURE 5.12
Heterochromatin Formation by RNAi
The RISC complex containing single-stranded siRNA can also recognize and bind to complementary
DNA sequences. When RISC associates with a repetitive DNA element, various histone modifying
enzymes and silencing complexes are activated to turn that region of DNA into heterochromatin. Once
this is done, the region is no longer transcribed into mRNA.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
13
FIGURE 5.13
Pathway of miRNA Inactivation of Gene Translation in Drosophila
Drosophila has several genes for miRNAs that control mRNA expression during development. The
genes are polycistronic, and an enzyme called Drosha cuts each of the hairpin structures to form premiRNAs. These exit the nucleus through nuclear pores. In the cytoplasm Dicer recognizes them and
cleaves the loop end. The resulting double-stranded miRNAs are recognized by RISC, separated into
single strands, and used to block translation or degrade complementary mRNA.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
14
FIGURE 5.14
Delivering dsRNA to C. elegans
(A) C. elegans can absorb dsRNA expressed in bacteria that they eat. (B) C. elegans can absorb
dsRNA by swimming in a solution containing dsRNA. (C) Injecting dsRNA into an egg will trigger gene
silencing in the developing worm.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
15
FIGURE 5.15
In Vitro Treatment with Dicer Generates siRNAs
The key to making siRNAs in vitro is cloning the target gene so that both the sense and antisense
strands are expressed into mRNA. The two strands anneal spontaneously, and when purified Dicer is
added, small siRNAs are produced.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
16
FIGURE 5.16
Design of shRNA Expression Vectors to Activate RNAi
Vectors can be designed to express different shRNA molecules. This retrovirus-based vector has two
complementary sequences about 20 nucleotides in length that form a stem, separated by a loop
region. When the vector is transformed into a cell, the shRNA is transcribed and activates gene
silencing.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
17
FIGURE 5.17
Constructing an RNAi Library
Each clone in the library must have two different promoters flanking the coding region. When the clone
is transcribed into mRNA, both an antisense and a sense transcript will be produced and the two
strands will come together to form double-stranded RNA.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
18
FIGURE 5.18
Making siRNA or shRNA Libraries
(A) Chemically synthesized oligonucleotides are cloned into a vector that has promoters to express both strands into
mRNA. When transcribed, the two strands pair together to form siRNA. (B) Restriction enzyme-generated siRNA
(REGS) creates a library of short hairpin RNA (shRNA). First the genes of interest are randomly digested with
restriction enzymes. Next, a 3’ hairpin is ligated to the pieces. MmeI then cuts the fragments to uniform lengths. MmeI
is a type I restriction enzyme that has its recognition sequence in the 3’ hairpin and its cutting site 20 nucleotides into
the restriction enzyme fragment. The fragments are next ligated to a 5’ hairpin and subjected to rolling-circle replication
to create a short hairpin coding region. The replicated linear fragment has repeating segments of 5’ hairpin (orange),
cloned fragment (gray), 3’ hairpin (blue), cloned fragment (gray), 5’ hairpin (orange), etc. Next, the repeating fragment
is cut with restriction enzymes that only recognize the 5’ hairpin (orange). The resulting piece has two complementary
sequences (orange and gray) that are separated by a loop (blue). When this is cloned and expressed into mRNA, the
two complementary sequences (orange and gray) anneal to form a short hairpin.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
19
FIGURE 5.19
Multiwell Plate Assays and Live Cell Microarrays
(A) Short-interfering RNA and short-hairpin RNA libraries can assess the function of mammalian
proteins. If the library is transfected into mammalian cells, each cell can be assessed for different
physical phenotypes, such as loss of adherence, mitotic arrest, or changed cell shape. The shRNA or
siRNA that causes an interesting phenotype can be isolated and sequenced to identify the protein that
was suppressed. (B) Rather than transforming cells, the siRNA or shRNA can be spotted onto
microscope slides. As cells grow and divide on the slide, the siRNAs are taken up by the cells and
initiate RNAi. The cells over each spot are assessed for a particular phenotype.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
20
A brief History
1982:Self-splicing in Tetrahymena pre-rRNA (group I intron)
Kruger et al, and Cech, Cell 31, 147-157 (1982)
1983:RNAse P is a ribozyme
Guerrier-Takada et al, and Altman, Cell, 35, 849-857 (1983)
Classes of Ribozymes
1. Group I tetrahymena
– Self-splicing
– Initiate splicing with a G nucleotide
– Uses a phosphoester transfer
mechanism
– Does not require ATP hydrolysis.
2. Group II self-splicing
– Initiate splicing with an internal A
– Uses a phosphoester transfer
mechanism
– Does not require ATP hydrolysis
3. Self-cleaving RNAs encoded by
viral genome to resolve the
concatameric molecules of the viral
genomic RNA produced
a. HDV ribozyme
b. Hairpin ribozyme
c. Hammerhead ribozyme
d. VS ribozyme
4. Rnase P
Group I and II introns
Mol. Biol. Genes, Fig. 13-9
A comparison of pre-mRNA, group I, and group II splicing mechanisms
FIGURE 5.20
Structure of the Twort Ribozyme
(A) Primary and secondary structure of the wild-type intron. The P1-P2 domain is highlighted in red,
the P3-P7 region is green, the P4-P6 domain is blue, the P9-P9.1 region is purple, the P7.1-P7.2
subdomain is yellow, and the product oligonucleotide is cyan. Dashed lines indicate key tertiary
structure contacts. Nucleotides in italics (P5a region) are disordered in the crystal. IGS, internal guide
sequence. (B) Ribbon diagram colored as in (A). The backbone ribbon is drawn through the
phosphate positions in the backbone. From: Golden, Kim, and Chase (2004). Crystal structure of a
phage Twort group I ribozyme-product complex. Reprinted by permission from: Macmillan
Publishers Ltd. Nat Struct Mol Biol12, 82–89, copyright 2005.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
27
FIGURE 5.21
Mechanism of Group I Self-Splicing Reaction
(A) The secondary structure of group I introns shows multiple hairpins that mediate the cleavage
reaction. In step I, a free guanosine (Red, G-OH) mediates the cleavage of the exon 1–intron
boundary. In step 2, the free end of exon 1 cleaves and ligates to exon 2. (B) Mechanism of group I
ribozyme cleavage. First the exon sequences are brought near the catalytic core via the internal guide
sequence (IGS). Exon 1 has an important uridine (U) that forms a U=G base pair with the IGS (dotted
line). The other end of the ribozyme has a binding site for the nucleophile, a free guanosine (Red),
which initiates intron removal by attacking the end of exon 1 with the 3’-OH of its ribose. The free 3’OH on the exon than reacts with the splice site on exon 2. The intron is spliced out and the two exons
are united (not shown). Although it appears that this reaction requires energy, the actual number of
bonds stays the same, and no net energy is needed.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
28
FIGURE 5.22
Group II Intron Splicing Reactions
The secondary and tertiary structure of group II introns brings the two exons together, but the reaction
mechanism does not require an external nucleophile. Instead, the 2’-OH of an internal conserved
adenine acts as a nucleophile, attacking the 5’ splice site and cleaving the phosphate backbone. The
3’ OH of the 5’ splice site attacks the 3’ splice site, resulting in two ligated exons and a free intron. The
intron forms a lariat structure.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
29
FIGURE 5.23
Life Cycle of Viroids
Viroids are single-stranded circular RNA genomes with no protein coat, but the ability to self-replicate.
First, the plus-stranded genome is converted into a concatemer of negative-stranded genomes with
rolling-circle replication. RNA polymerase converts the negative-stranded genomes into plus-stranded
genomes, which are separated and ligated into circular genomes. The hammerhead ribozyme,
embedded within the viroid genome, catalyzes the separation and ligation into a circle.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
30
Hammerhead ribozymes
• A 58 nt structure is used in self-cleavage
• The sequence CUGA adjacent to stemloops is sufficient for cleavage
5'
3'
AA
A
GGCC
CCGG A
CG
U A
C G
AUC
U
G
GU A
Bond that is cleaved.
ACCAC
C UGGUG
CUGA is required for catalysis
The hairpin ribozyme (plant virus)
From Lilley TIBS
(2003)
FIGURE 5.24
Secondary Structure of Hairpin Ribozyme
The minus strand of the tobacco ring spot virus genome is shown with the cleavage site indicated by
the red arrow.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
33
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
34
FIGURE 5.25
Antisense Construct with Ribozyme Inactivates Target mRNA
The chimeric antisense ribozyme not only has the ability to bind to a specific target mRNA, but also
cleaves the target mRNA. A traditional antisense oligonucleotide must rely on recruiting RNase H to
digest the target mRNA. However, RNase H also degrades the antisense oligonucleotide, which
cannot therefore be reused.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
35
FIGURE 5.26
Nuclease Resistant Ribozyme Bound to a Target mRNA
The ribozyme consists of 2’-O-methyl nucleotides and phosphorothioate linkages. At the 3’-end, a 3’-3’
deoxyabasic sugar (iB) is added. All three modifications prevent nuclease degradation of the ribozyme.
The five green nucleotides (rA or rG) form the catalytic core and cut the target mRNA at the cleavage
site. The H at the cleavage site represents an A, C, or U.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
36
SELEX :Systematic Evolution
of Ligands by EXponential
enrichment
A brief History
Ellington and Szostak, Nature (1990)
Tuerk and Gold , Science (1990)
Wilson and Szostak, Ann.Rev.Bioc. (1999)
FIGURE 5.27
RNA SELEX Identifies New Ribozyme Substrates
The key to RNA SELEX is to create a very large pool of random RNA sequences. First, DNA
oligonucleotides are chemically synthesized to create a large pool of random sequences. These are
converted into double-stranded DNA with a primer and Klenow polymerase. The primer adds an RNA
polymerase binding site. The dsDNA oligonucleotides are transcribed into RNA with RNA polymerase.
This large pool of random RNA is then screened for binding to the ribozyme. Any RNA molecules that
bind are kept, and the rest are discarded. Those that bind are converted to cDNA and amplified with
PCR. The selection process can be repeated numerous times to enrich for RNA sequences that bind.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
39
The ATP aptamer
structure
- Selection against small molecules
- Selection against proteins
- Selection of new ribozymes (RNA world
FIGURE 5.28
In Vitro Selection of Ribozyme Ligation
The pink molecule represents the large pool of random RNA sequences. At the 5’ end, there is a
substrate sequence with a terminal triphosphate. At the 3’ end, a blue effector molecule is bound to
facilitate ligation. The second substrate (purple) is then incubated with the random pool of
oligonucleotides. If any of the random sequences catalyze the ligation of the substrates, the resulting
species will be larger and may be separated out by gel electrophoresis. The ligated oligonucleotide is
isolated from the gel, amplified by PCR, and finally sequenced.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
42
FIGURE 5.29
In Vitro Evolution of Ribozymes
In vitro evolution tries to find an RNA sequence that works as a ribozyme. In this example, the
researcher is looking for a ribozyme that catalyzes the addition of a metal ion (M+) to a porphyrin ring.
The pool of random RNA sequences is created and amplified with error-prone PCR to increase the
odds of finding one or two sequences that catalyze the reaction. Each successive round of selection
and mutation improves any ribozymes that are found.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
43
FIGURE 5.31
Riboswitches Control mRNA Expression
Riboswitches alternate between two stem and loop structures depending on the presence or absence
of the signal metabolite. (A) In the attenuation mechanism, the presence of the signal metabolite
results in formation of the terminator structure and transcription is aborted. (B) In the translational
inhibition mechanism, the presence of the metabolite results in sequestration of the Shine-Dalgarno
sequence, which prevents translation of the mRNA.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
44
FIGURE 5.32
Ribozyme Riboswitch of B. subtilis GlmS Gene
(A) Cell wall synthesis occurs during growth conditions. When the cell is growing, levels of UDPGlcNAc are low and are quickly converted into cell wall components. (B) If the cell is not growing,
UDP-GlcNAc is not incorporated into the cell wall and accumulates. The excess UDP-GlcNAc binds to
a riboswitch on the glmS gene. Once bound, it activates the self-cleaving ribozyme to degrade the
mRNA, and halts the production of glutamine fructose 6-phosphate amidotransferase.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
45
FIGURE 5.33
Designing Allosteric Ribozymes
(A) Modular design of a ribozyme. The ribozyme has three different domains joined together. The
substrate domain (light green background) base-pairs with the ribosyme domain (light purple
background), and the aptamer domain binds the allosteric effector (ATP in this example). (B) In vitro
selection scheme to identify ribozymes that are active only when bound to an effector (i.e., are
allosteric). First, all ribozymes that catalyze substrate cleavage without an effector are removed. If the
substrate is cleaved without the effector, the ribozyme will move faster during electrophoresis. Only
the uncleaved ribozyme/substrate band is isolated from the gel. Next, the ribozymes are mixed with an
effector molecule. This time, the ribozymes that cleave the substrate are isolated. Repeated cycles of
isolation will identify a ribozyme that works only with the effector.
Companion site for Biotechnology. by Clark
Copyright © 2009 by Academic Press. All rights reserved.
46