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and ES cells, the resulting tetraploid cells emphasize the limitations of reprogram- widely adopted and useful tool for the analy-
containing thymocyte-derived nuclei dis- ming cell lineages without understanding sis of loss-of-function phenotypes that
played a growth morphology and behavior the underlying biological processes. develop over longer time periods4–8.
indistinguishable from those of ES cells5. In The Håkelien assay involves extensive cell Molecules of siRNA are 21- to 23-
the assay of Håkelien et al., several obstacles manipulation to induce reprogramming. nucleotide RNAs, with characteristic 2- to 3-
(such as size, stability, nuclear import, and Consequently, the applicability of this tech- nucleotide 3′-overhanging ends resembling
time) may limit delivery of extract proteins nology in producing reprogrammed cell the RNAse III processing products of long
to the target nucleus and therefore limit lines for therapeutic purposes remains double-stranded RNAs (dsRNAs) that nor-
nuclear reprogramming. For example, it is undetermined. A large amount of additional mally initiate RNAi. When introduced into a
possible that the modifications observed in data are required before such a system could cell, they assemble with yet-to-be-identified
© 2002 Nature Publishing Group

the reprogrammed fibroblasts were initiated be applied to the generation of stem cells to proteins of an endonuclease complex (RNA-
by dominant transcription factor(s) present be used directly in cell replacement therapies induced silencing complex), which then
in the protein extract and that, given expo- for human patients. guides target mRNA cleavage. As a conse-
sure to a greater complexity of factors, more Setting these reservations aside, the quence of degradation of the targeted
extensive reprogramming would result. authors’ assay provides a potentially power- mRNA, cells with a specific phenotype char-
Recent experiments have demonstrated that ful system for analyzing nuclear reprogram- acteristic of suppression of the correspond-
overexpression of the transcription factor ming events as they occur in vitro. It will be ing protein product are obtained. The small
Msx1 can initiate dedifferentiation of multi- exciting to apply similar methodologies to size of siRNAs, compared with traditional
nucleated myotubes to a more plastic single the analysis of other systems, including ES antisense molecules, prevents activation of
nucleated precursor cell type11. Further cells, EG cells, and various cancer cell types. the dsRNA-inducible interferon system pre-
experiments using the reprogramming tech- Such analyses may allow identification of sent in mammalian cells. This avoids the
nology of Håkelien et al. should clarify these molecules central to biological processes as nonspecific phenotypes normally produced
issues. diverse as the establishment of pluripotency, by dsRNA larger than 30 base pairs in somat-
Several laboratories have reported the the control of cell dedifferentiation, and the ic cells.
transdifferentiation of stem cells to tissues onset of cancer. Intracellular transcription of small RNA
outside their normal developmental lin- molecules can be achieved by cloning the
eage. For instance, hematopoietic stem siRNA templates into RNA polymerase III
1. Håkelien, A.M. et al. Nature Biotechnology 20,
cells can apparently give rise to cells of the 460–466 (2002). (Pol III) transcription units, which normally
liver and central nervous system, whereas 2. Landsverk, H.B. et al. EMBO J. 3, 384–389 (2002). encode the small nuclear RNA (snRNA) U6
3. Kikyo, N. et al. Science 289, 2360–2362 (2000).
neuronal stem cells can change into 4. Rideout, W.M., 3rd et al. Science 293, 1093–1098
or the human RNAse P RNA H1. Two
hematopoietic cells9,10. However, recent (2001). approaches have been developed for express-
experiments indicate that the isolation and 5. Tada, M. et al. Curr. Biol. 11, 1553–1558 (2001). ing siRNAs: in the first, sense and antisense
6. Terada, N. et al. Nature 416, 542–545 (2002).
culture of adult stem cells or cell fusion 7. Ying, Q., Nichols, J., Evans, E.P. & Smith, A.G.
strands constituting the siRNA duplex are
events may explain the unexpectedly high Nature 416, 545–548 (2002). transcribed by individual promoters (Fig.
level of plasticity of adult stem cells6–8. 8. Morshead, C.M. et al. Nat. Med. 8, 268–273 (2002). 1A)5,6; in the second, siRNAs are expressed as
9. Lagasse, E. et al. Nat. Med 6, 1229–1234 (2000).
These observations underscore how poorly 10. Krause, D.S. et al. Cell 105, 369–377 (2001). fold-back stem–loop structures that give rise
cellular plasticity is understood and 11. Odelberg, S.J. et al. Cell 103, 1099–1109 (2000). to siRNAs after intracellular processing (Fig.
1B)4,7,8. The endogenous expression of
siRNAs from introduced DNA templates is
thought to overcome some limitations of
Expanding small RNA interference exogenous siRNA delivery, in particular the
transient loss of phenotype.
U6 and H1 RNA promoters are members
Several reports describe vector systems capable of producing small of the type III class of Pol III promoters9.
interfering RNAs for downregulating gene expression in These promoters are unusual in that almost
all their elements, with the exception of the
mammalian cells. first transcribed nucleotide (+1 position),
are located upstream of the transcribed
Thomas Tuschl region, so that almost any inserted sequence
shorter than 400 nucleotides can be tran-
Small interfering RNAs (siRNAs) are power- experiments have been the transient nature scribed. They are therefore ideally suited to
ful sequence-specific reagents designed to of siRNA transfer into cells (achieved by the expression of ∼21-nucleotide siRNAs or
suppress the expression of genes in cultured such classic methods as liposome-mediated ∼50- nucleotide RNA stem–loops. The U6
mammalian cells through a process known transfection, electroporation, or microinjec- promoter and the H1 promoter are different
as RNA interference (RNAi)1–3. Until recent- tion) and the requirement for chemical or in size but contain the same conserved
ly, two limitations of siRNA gene-targeting enzymatic synthesis of siRNAs before appli- sequence elements or protein-binding
cation. In this issue and in Science and Genes sites10. The +1 nucleotide of the U6-like pro-
& Development, several independent res- moters is always guanosine, whereas it is
Thomas Tuschl is an EMBO young earch groups now describe alternative adenosine for H1. Interestingly, changing the
investigator, Department of Cellular approaches for producing cells in which spe- +1 adenosine to uridine, cytidine, or guano-
Biochemistry, Max Planck Institute for cific genes have been targeted by intracellu- sine within H1-expressed stem–loop
Biophysical Chemistry, Am Fassberg 11, D- lar expression of siRNAs from plasmid DNA sequences does not seem to affect gene
37077 Göttingen, Germany in an attempt to address these limitations. silencing, indicating that H1 promoters may
( The data indicate that siRNA will become a be more flexible than U6 promoters in

446 nature biotechnology • VOLUME 20 • MAY 2002 •


A exogenous delivery of RNA hairpins may

+1 +1 represent an alternative to exogenous deliv-
ery of siRNA duplexes, as enzymatic RNA
U6 promoter GN17C TTTTT U6 promoter GN'17C TTTTT hairpin synthesis would require only a single
DNA template8. Intracellular processing of
Sense Pol III, Antisense
hairpin RNAs appears to require Dicer
hybridization RNase III because Dicer suppressed cells do
not support hairpin-mediated target gene
5'-GN17 CU 1–4 Suppression of protein levels by exoge-
© 2002 Nature Publishing Group

siRNA duplex nous siRNAs is transient: levels of the target-

ed protein in siRNA-treated cells typically
1–4 UCN'17 G-5' recover between five and seven days after
Select target sequence -AGN17C- siRNA transfection, that is, after seven to
ten rounds of cell division (T.T. et al.,
unpublished observations). In three of the
B +1 new studies, the periods of persistent sup-
pression or stable loss-of-function pheno-
H1 pr. AN 17-TT-loop-N'18T T T T T type were extended by producing stable cell
lines propagating the siRNA expression
cassettes. Miyagishi and Taira6 suppressed
β-catenin, a protein involved in cadherin-
mediated cell–cell adhesion, for over one
week. The β-catenin-targeting siRNA
5'-AN18 UU strands were expressed from a plasmid
Short hairpin RNA containing the Epstein–Barr virus DNA
replication origin, and the plasmid was
1–4 UN'18 propagated in cells stably expressing the
Epstein–Barr virus nuclear antigen 1
Processing (EBNA-1). Brummelkamp et al.4 and
Paddison et al.8 produced cells that stably
5'-AN18 UU suppressed p53, an important protein in
the cellular response to DNA damage.
siRNA duplex
Silencing of p53 was observed for over two
1–4 UN'18 -5' months in antibiotic-selected, stably trans-
© Bob Crimi

fected cells, indicating that long-term

expression of siRNAs is not toxic to cells.
Select target sequence -AAN18- However, most of the applications of
siRNA expression systems described in
Figure 1. Endogenous expression of siRNAs. (A) Expression cassette for sense and antisense these four papers focused on transient
siRNAs using the U6 snRNA promoter5,6. The 250 bp U6 snRNA promoter is illustrated as a yellow transfection of siRNA expression vectors
box, the Pol III terminator signal composed of a run of thymidines is shown as a light purple box, and
the spacer between the sense and antisense (indicated with a prime symbol) expression element is and on the analysis of phenotypes associat-
shown as a blue box. The siRNA elements are highlighted in pink. The target site, preferably selected ed with protein suppression within a few
for optimal vector design, is indicated at the bottom. (B) H1 RNA–based Pol III cassette for days after transfection, and therefore did
expressing hairpin RNAs that are subsequently processed to siRNAs4. The H1 RNA Pol III promoter not assess the effects of siRNA expression
is only 100 bp in size, but contains all the essential sequence motifs present in the U6 snRNA
promoter10. Hairpin RNAs with gene silencing properties were also obtained by using a U6 promoter8. beyond the temporal window of silencing
In this case, transcript synthesis was initiated with a +1 guanosine and the 3′ end of the sense strand by the exogenous siRNA transfection
was joined by a loop with the antisense strand. approach.
Transfection of plasmid DNA, rather
than synthetic siRNAs, may appear advan-
regard to +1 sequence changes or may be stem–loop sequences with 3′-overhanging tageous, considering the danger of RNAse
able to initiate transcription at the first uridines were as effective at silencing target contamination and the costs of chemically
downstream purine nucleotide encoded by genes as were the synthetic siRNAs4,7, and synthesized siRNAs or siRNA transcription
the template DNA. RNA transcription is ter- that even blunt-ended duplexes with up to kits. For practical applications, however,
minated when Pol III encounters a run of 29 base pairs were able to mediate RNAi in the considerable time invested in preparing
four or five thymidines after incorporation cultured cells8. Although the size, orienta- and amplifying siRNA expression vectors,
of several uridine residues10. tion, and sequence of the loop appear to be and the transfection efficiency of plasmids
As was expected from previous analysis of important, endogenously expressed RNA relative to synthetic siRNAs, must also be
RNAi, co-expression of sense and antisense hairpins connected by the 3′ end of the sense considered. Furthermore, targeting of
siRNAs mediated silencing of target genes, strand and the 5′ end of the siRNA antisense essential genes causes arrest in cell growth
whereas expression of sense or antisense strand by a 9-nucleotide loop sequence or cell death within one to three days after
siRNA alone did not greatly affect target were so rapidly processed to siRNAs that delivery of siRNAs, and thus in many
gene expression5–7. More surprising was the the 49-nucleotide precursor was barely instances long-term silencing is unneces-
finding that DNA constructs encoding 19-bp detectable4. This finding also suggests that sary; however, the development of • MAY 2002 • VOLUME 20 • nature biotechnology 447


inducible siRNA expression systems may Incorporation of siRNA expression cas- detection by sandwich enzyme-linked
provide an interesting alternative in such settes into retroviral vectors may also allow immunosorbent assays (ELISAs) or west-
cases6. For example, cells could be grown the targeting of primary cells previously ern blotting. Though sufficient for most
on a large scale before induction of the refractory to siRNA or plasmid DNA trans- standard applications, these techniques are
knockdown, which may be beneficial for fection8. Considering the high specificity of not sensitive enough to meet the increas-
proteomic analysis. Nonetheless, in target- siRNAs4–7, the approach should allow the ingly stringent detection limits required in
ing nonessential proteins, stable “knock- targeting of disease-derived transcripts the postgenomics era. For example, many
down” cells may be of great value when with point mutations, such as RAS or TP53 proteins, such as cytokines and certain
studying inducible processes such as UV or oncogene transcripts, without alteration of kinases, exert their functions at concentra-
other irradiation damage response, the remaining wild-type allele. Finally, tions considerably lower than the thresh-
© 2002 Nature Publishing Group

host–pathogen interactions, or cell differ- because of the automation developed for olds of detection attainable by these
entiation. high-throughput sequence analysis of the methods. To overcome these limitations,
Considering all the pros and cons of various genomes, the DNA-based methodol- variations of the sandwich ELISA with
expressed versus synthetic siRNAs, it is ogy may also be a cost-effective alternative for markedly increased sensitivity, such as the
probably most effective to begin the search automated genome-wide loss-of-function “immuno–polymerase chain reaction”
for highly effective siRNAs with synthetic, phenotypic analysis, especially when com- (immuno-PCR)2 and a technique termed
ready-to-use duplex RNAs of defined bined with miniaturized array-based pheno- “immunodetection amplified by T7 RNA
sequence and length, and to select the syn- typic screens11. polymerase” (IDAT)3, have been devel-
thetic sequences such that they are readily oped. These methods combine antigen
compatible with the sequence require- 1. Elbashir, S.M. et al. Nature 411, 494–498 (2001).
recognition by a biotin-tagged antibody
ments for expression within U6 or H1 RNA 2. Caplen, N.J. et al. Proc. Natl. Acad. Sci. USA 98, with PCR amplification of a biotinylated
expression cassettes. This will first entail 9742–9747 (2001). reporter DNA, linking the two to one
3. Harborth, J. et al. J. Cell Sci. 114, 4557–4565
ensuring that in the U6 snRNA and H1 (2001). another via a streptavidin molecule. Tags
RNA systems that the +1 position is a 4. Brummelkamp, T.R. et al. Science 21 March 2002 other than biotin, alternative bridging
guanosine7,9 and an adenosine, respective- 5. Lee, N.S. et al. Nat. Biotechnol. 20, 500–505 (2002).
moieties, or covalent coupling methods
ly. In addition, it will require that uridines 6. Miyagishi, M. & Taira, K. Nat. Biotechnol. 20, have also been used in immuno-PCR
be present in the 3′-terminal position 497–500 (2002). experiments. When performed under “real-
7. Paul, C.P. et al. Nat. Biotechnol. 20, 505–508 (2002).
encoded by the oligothymidine Pol III ter- 8. Paddison, P.J. et al. Genes & Dev. 16, in press time PCR” conditions, in which a fluori-
minator signal sequence9. (2002). genic oligonucleotide probe is used to mea-
9. Paule, M.R. & White, R.J. Nucleic Acids Res. 28,
Ultimately, the possibility of stable 1283–1298 (2000). sure PCR product accumulation, these
expression of siRNAs may pave the road for 10. Myslinski, E. et al. Nucleic Acids Res. 29, methods can provide very accurate and
2502–2509 (2001).
new gene therapy applications, such as 11. Ziauddin, J. & Sabatini, D.M. Nature 411, 107–110
highly sensitive quantification of the PCR
treatment of persistent viral infections. (2001). products.
Fredrickson et al. do not use any anti-
bodies, tags, or linker molecules and yet
achieve levels of protein detection compet-
itive with those of immuno-PCR. They
Bringing picomolar protein detection have used a clever strategy whereby an
“antigen” is detected by nucleic acid–based
into proximity receptor molecules, so-called aptamers4. As
a test case, the researchers use a pair of
DNA aptamers that binds to the homod-
Combining target recognition by two aptamers, enzymatic ligation, imer of the target analyte protein, platelet-
and PCR, the proximity ligation method enables the detection of derived growth factor B-chain (PDGF-BB).
minute amounts of proteins. Each aptamer has a different DNA-
sequence extension that does not interfere
with its folding and is not required for tar-
Michael Famulok get recognition. Binding of the aptamer
pair brings the ends of the oligonucleotide
Thousands of proteins with potential diag- pathogens. The ability to monitor slight extensions into close spatial proximity
nostic and/or therapeutic applications are differences in the amounts of proteins and (Fig. 1A) so that a “splint” oligonucleotide
expected to emerge from the various other biomolecules within the smallest can hybridize to both ends, which are sub-
genome projects. Proteins in different tis- possible detection volumes down to the sequently ligated together by T4 DNA lig-
sues or individual cells can be up- or single-cell level is of utmost importance ase. The ligated species can then act as a
downregulated in response to internal or not only for proteomics research but also PCR template and the amplified PCR
external stimuli, signal transduction, tran- for diagnostic and technological purposes. product can be monitored and quantified
scriptional control, medication, disease, or In this issue, Fredriksson et al.1 describe a under real-time PCR conditions, whereas
new method, proximity ligation, that no signal is obtained with unligated probes
allows the detection and quantification of (Fig. 1B).
Michael Famulok is a professor at the Kekulé minute amounts of a specific protein. The sensitivity that can be achieved with
Institute of Organic Chemistry and Methods currently in widespread use for the method is remarkable. As few as 24,000
Biochemistry, Universität Bonn, Gerhard- research requiring standard protein detection molecules, or 4 × 10–20 moles, of the PDGF-
Domagk-Strasse 1, 53121 Bonn, Germany include two-dimensional gel electrophoresis, BB protein could be detected, approxi-
( mass spectrometry, and antibody-based mately 1,000-fold fewer than could be

448 nature biotechnology • VOLUME 20 • MAY 2002 •