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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Deep Sequencing Analyses of DsiRNAs Reveal the Influence of 3′ Terminal Overhangs
on Dicing Polarity, Strand Selectivity, and RNA Editing of siRNAs</title><author><name sortKey="Zhou, Jiehua" sort="Zhou, Jiehua" uniqKey="Zhou J" first="Jiehua" last="Zhou">Jiehua Zhou</name><affiliation><nlm:aff id="aff1"><institution>Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Song, Min Sun" sort="Song, Min Sun" uniqKey="Song M" first="Min-Sun" last="Song">Min-Sun Song</name><affiliation><nlm:aff id="aff1"><institution>Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Jacobi, Ashley M" sort="Jacobi, Ashley M" uniqKey="Jacobi A" first="Ashley M" last="Jacobi">Ashley M. Jacobi</name><affiliation><nlm:aff id="aff2"><institution>Integrated DNA Technologies, Inc.</institution> Coralville, Iowa,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Behlke, Mark A" sort="Behlke, Mark A" uniqKey="Behlke M" first="Mark A" last="Behlke">Mark A. Behlke</name><affiliation><nlm:aff id="aff2"><institution>Integrated DNA Technologies, Inc.</institution> Coralville, Iowa,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Wu, Xiwei" sort="Wu, Xiwei" uniqKey="Wu X" first="Xiwei" last="Wu">Xiwei Wu</name><affiliation><nlm:aff id="aff3"><institution>Department of Molecular Medicine, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Rossi, John J" sort="Rossi, John J" uniqKey="Rossi J" first="John J" last="Rossi">John J. Rossi</name><affiliation><nlm:aff id="aff1"><institution>Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff4"><institution>Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">23343928</idno><idno type="pmc">3384246</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384246</idno><idno type="RBID">PMC:3384246</idno><idno type="doi">10.1038/mtna.2012.6</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">000004</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000004</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Deep Sequencing Analyses of DsiRNAs Reveal the Influence of 3′ Terminal Overhangs
on Dicing Polarity, Strand Selectivity, and RNA Editing of siRNAs</title><author><name sortKey="Zhou, Jiehua" sort="Zhou, Jiehua" uniqKey="Zhou J" first="Jiehua" last="Zhou">Jiehua Zhou</name><affiliation><nlm:aff id="aff1"><institution>Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Song, Min Sun" sort="Song, Min Sun" uniqKey="Song M" first="Min-Sun" last="Song">Min-Sun Song</name><affiliation><nlm:aff id="aff1"><institution>Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Jacobi, Ashley M" sort="Jacobi, Ashley M" uniqKey="Jacobi A" first="Ashley M" last="Jacobi">Ashley M. Jacobi</name><affiliation><nlm:aff id="aff2"><institution>Integrated DNA Technologies, Inc.</institution> Coralville, Iowa,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Behlke, Mark A" sort="Behlke, Mark A" uniqKey="Behlke M" first="Mark A" last="Behlke">Mark A. Behlke</name><affiliation><nlm:aff id="aff2"><institution>Integrated DNA Technologies, Inc.</institution> Coralville, Iowa,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Wu, Xiwei" sort="Wu, Xiwei" uniqKey="Wu X" first="Xiwei" last="Wu">Xiwei Wu</name><affiliation><nlm:aff id="aff3"><institution>Department of Molecular Medicine, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Rossi, John J" sort="Rossi, John J" uniqKey="Rossi J" first="John J" last="Rossi">John J. Rossi</name><affiliation><nlm:aff id="aff1"><institution>Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff4"><institution>Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></nlm:aff></affiliation></author></analytic><series><title level="j">Molecular therapy. Nucleic acids</title><idno type="eISSN">2162-2531</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>25/27 Base duplex RNAs that are substrates for Dicer have been demonstrated to enhance
RNA interference (RNAi) potency and efficacy. Since the target sites are not always
equally susceptible to suppression by small interfering RNA (siRNA), not all 27-mer
duplexes that are processed into the corresponding conventional siRNAs show increased
potency. Thus random designing of Dicer-substrate siRNAs (DsiRNAs) may generate siRNAs
with poor RNAi due to unpredictable Dicer processing. Previous studies have demonstrated
that the 3<bold>′</bold>-overhang affects dicing cleavage site and the orientation of
Dicer entry. Moreover, an asymmetric 27-mer duplex having a 3<bold>′</bold> two-nucleotide
overhang and 3<bold>′</bold>-DNA residues on the blunt end has been rationally designed to
obtain greater efficacy. This asymmetric structure directs dicing to predictably yield a
single primary cleavage product. In the present study, we analyzed the <italic>in vitro</italic>and intracellular dicing patterns of chemically synthesized duplex RNAs with different
3<bold>′</bold>-overhangs. Consistent with previous studies, we observed that Dicer
preferentially processes these RNAs at a site 21–22 nucleotide (nt) from the
two-base 3<bold>′</bold>-overhangs. We also observed that the direction and ability of
human Dicer to generate siRNAs can be partially or completely blocked by DNA residues at
the 3<bold>′</bold>-termimi. To examine the effects of various 3<bold>′</bold>-end
modifications on Dicer processing in cells, we employed Illumina Deep sequencing analyses
to unravel the fates of the asymmetric 27-mer duplexes. To validate the strand selection
process and knockdown capabilities we also conducted dual-luciferase psiCHECK reporter
assays to monitor the RNAi potencies of both the “sense” (S) and
“antisense” (AS) strands derived from these DsiRNAs. Consistent with our
<italic>in vitro</italic> Dicer assays, the asymmetric duplexes were predictably processed into
desired primary cleavage products of 21–22-mers in cells. We also observed the
trimming of the 3<bold>′</bold> end, especially when DNA residues were incorporated into
the overhangs and this trimming ultimately influenced the Dicer-cleavage site and RNAi
potency. Moreover, the observation that the most efficacious strand was the most abundant
revealed that the relative frequencies of each “S” or “AS” strand
are highly correlated with the silencing activity and strand selectivity. Collectively,
our data demonstrate that even though the only differences between a family of DsiRNAs was
the 3<bold>′</bold> two-nuclotide overhang, dicing polarity and strand selectivity are
distinct depending upon the sequence and chemical nature of this overhang. Thus, it is
possible to predictably control dicing polarity and strand selectivity <italic>via</italic> simply
changing the 3<bold>′</bold>-end overhangs without altering the original duplex sequence.
These optimal design features of 3<bold>′</bold>-overhangs might provide a facile approach
for rationally designing highly potent 25/27-mer DsiRNAs.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Fire, A" uniqKey="Fire A">A Fire</name></author><author><name sortKey="Xu, S" uniqKey="Xu S">S Xu</name></author><author><name sortKey="Montgomery, Mk" uniqKey="Montgomery M">MK Montgomery</name></author><author><name sortKey="Kostas, Sa" uniqKey="Kostas S">SA Kostas</name></author><author><name sortKey="Driver, Se" uniqKey="Driver S">SE Driver</name></author><author><name sortKey="Mello, Cc" uniqKey="Mello C">CC Mello</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zamore, Pd" uniqKey="Zamore P">PD Zamore</name></author><author><name sortKey="Tuschl, T" uniqKey="Tuschl T">T Tuschl</name></author><author><name sortKey="Sharp, Pa" uniqKey="Sharp P">PA Sharp</name></author><author><name sortKey="Bartel, Dp" uniqKey="Bartel D">DP 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uniqKey="Martin D">DM Martin</name></author><author><name sortKey="Clamp, M" uniqKey="Clamp M">M Clamp</name></author><author><name sortKey="Barton, Gj" uniqKey="Barton G">GJ Barton</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Mol Ther Nucleic Acids</journal-id><journal-id journal-id-type="iso-abbrev">Mol Ther Nucleic Acids</journal-id><journal-title-group><journal-title>Molecular therapy. Nucleic acids</journal-title></journal-title-group><issn pub-type="epub">2162-2531</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">23343928</article-id><article-id pub-id-type="pmc">3384246</article-id><article-id pub-id-type="pii">mtna20126</article-id><article-id pub-id-type="doi">10.1038/mtna.2012.6</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Deep Sequencing Analyses of DsiRNAs Reveal the Influence of 3′ Terminal Overhangs
on Dicing Polarity, Strand Selectivity, and RNA Editing of siRNAs</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Zhou</surname><given-names>Jiehua</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Song</surname><given-names>Min-Sun</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Jacobi</surname><given-names>Ashley M</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Behlke</surname><given-names>Mark A</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Xiwei</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Rossi</surname><given-names>John J</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff4">4</xref></contrib><aff id="aff1"><label>1</label><institution>Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></aff><aff id="aff2"><label>2</label><institution>Integrated DNA Technologies, Inc.</institution> Coralville, Iowa,<country>USA</country></aff><aff id="aff3"><label>3</label><institution>Department of Molecular Medicine, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></aff><aff id="aff4"><label>4</label><institution>Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope</institution>, Duarte, California,<country>USA</country></aff></contrib-group><author-notes><corresp id="caf1"><label>*</label>Department of Molecular and Cellular Biology, Beckman Research
Institute of City of Hope, 1500 East Duarte Road, Duarte, California 91010, USA. E-mail
address: <email>jrossi@coh.org</email></corresp></author-notes><pub-date pub-type="ppub"><month>04</month><year>2012</year></pub-date><pub-date pub-type="epub"><day>03</day><month>04</month><year>2012</year></pub-date><pub-date pub-type="pmc-release"><day>1</day><month>4</month><year>2012</year></pub-date><volume>1</volume><issue>4</issue><fpage>e17</fpage><lpage></lpage><history><date date-type="received"><day>23</day><month>01</month><year>2012</year></date><date date-type="rev-recd"><day>18</day><month>02</month><year>2012</year></date><date date-type="accepted"><day>03</day><month>04</month><year>2012</year></date></history><permissions><copyright-statement>Copyright © 2012 American Society of Gene & Cell Therapy</copyright-statement><copyright-year>2012</copyright-year><copyright-holder>American Society of Gene & Cell Therapy</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc-nd/3.0/"><pmc-comment>author-paid</pmc-comment>
<license-p><italic>Molecular Therapy-Nucleic Acids</italic> is an open-access journal published
by Nature Publishing Group. This work is licensed under the Creative Commons
Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this
license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/</license-p></license></permissions><abstract><p>25/27 Base duplex RNAs that are substrates for Dicer have been demonstrated to enhance
RNA interference (RNAi) potency and efficacy. Since the target sites are not always
equally susceptible to suppression by small interfering RNA (siRNA), not all 27-mer
duplexes that are processed into the corresponding conventional siRNAs show increased
potency. Thus random designing of Dicer-substrate siRNAs (DsiRNAs) may generate siRNAs
with poor RNAi due to unpredictable Dicer processing. Previous studies have demonstrated
that the 3<bold>′</bold>-overhang affects dicing cleavage site and the orientation of
Dicer entry. Moreover, an asymmetric 27-mer duplex having a 3<bold>′</bold> two-nucleotide
overhang and 3<bold>′</bold>-DNA residues on the blunt end has been rationally designed to
obtain greater efficacy. This asymmetric structure directs dicing to predictably yield a
single primary cleavage product. In the present study, we analyzed the <italic>in vitro</italic>and intracellular dicing patterns of chemically synthesized duplex RNAs with different
3<bold>′</bold>-overhangs. Consistent with previous studies, we observed that Dicer
preferentially processes these RNAs at a site 21–22 nucleotide (nt) from the
two-base 3<bold>′</bold>-overhangs. We also observed that the direction and ability of
human Dicer to generate siRNAs can be partially or completely blocked by DNA residues at
the 3<bold>′</bold>-termimi. To examine the effects of various 3<bold>′</bold>-end
modifications on Dicer processing in cells, we employed Illumina Deep sequencing analyses
to unravel the fates of the asymmetric 27-mer duplexes. To validate the strand selection
process and knockdown capabilities we also conducted dual-luciferase psiCHECK reporter
assays to monitor the RNAi potencies of both the “sense” (S) and
“antisense” (AS) strands derived from these DsiRNAs. Consistent with our
<italic>in vitro</italic> Dicer assays, the asymmetric duplexes were predictably processed into
desired primary cleavage products of 21–22-mers in cells. We also observed the
trimming of the 3<bold>′</bold> end, especially when DNA residues were incorporated into
the overhangs and this trimming ultimately influenced the Dicer-cleavage site and RNAi
potency. Moreover, the observation that the most efficacious strand was the most abundant
revealed that the relative frequencies of each “S” or “AS” strand
are highly correlated with the silencing activity and strand selectivity. Collectively,
our data demonstrate that even though the only differences between a family of DsiRNAs was
the 3<bold>′</bold> two-nuclotide overhang, dicing polarity and strand selectivity are
distinct depending upon the sequence and chemical nature of this overhang. Thus, it is
possible to predictably control dicing polarity and strand selectivity <italic>via</italic> simply
changing the 3<bold>′</bold>-end overhangs without altering the original duplex sequence.
These optimal design features of 3<bold>′</bold>-overhangs might provide a facile approach
for rationally designing highly potent 25/27-mer DsiRNAs.</p></abstract><kwd-group><kwd>RNA interference</kwd><kwd>Dicer-substrate siRNA (DsiRNA)</kwd><kwd>Deep sequencing</kwd><kwd>3′-overhang</kwd><kwd>Dicing polarity</kwd></kwd-group></article-meta></front><body><sec><title>Introduction</title><p>RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing
process triggered by 21–25 nucleotide (nt) small interfering RNAs (siRNAs). In cells
these siRNAs are generated by the ribonuclease III Dicer which processes these siRNAs from
longer double-stranded RNAs.<sup><xref ref-type="bibr" rid="bib1">1</xref>,<xref ref-type="bibr" rid="bib2">2</xref></sup> In association with Dicer, the cleaved small RNA products
possessing a 5′-phosphate and 2-base 3′ overhang are loaded into large
multiprotein complexes termed RNA-induced silencing complexes (RISC) and one of the two
strands is selected as a guide for the sequence-specific silencing of the complementary
target RNA.<sup><xref ref-type="bibr" rid="bib2">2</xref>,<xref ref-type="bibr" rid="bib3">3</xref>,<xref ref-type="bibr" rid="bib4">4</xref>,<xref ref-type="bibr" rid="bib5">5</xref></sup> The PAZ domain, an
RNA-binding domain of Dicer which is also found in Argonaute proteins, specifically
recognizes the 3′ end of single-stranded RNA, suggesting it can function as a module
for anchoring the 3′ end of the guide strand within the RISC.<sup><xref ref-type="bibr" rid="bib5">5</xref>,<xref ref-type="bibr" rid="bib6">6</xref></sup> For the Dicer-substrate
siRNAs (DsiRNAs), the 3′ overhang therefore<sup><xref ref-type="bibr" rid="bib7">7</xref>,<xref ref-type="bibr" rid="bib8">8</xref>,<xref ref-type="bibr" rid="bib9">9</xref></sup>affects dicing polarity (binding of Dicer) as well as subsequent strand selectivity in
RISC,<sup><xref ref-type="bibr" rid="bib10">10</xref>,<xref ref-type="bibr" rid="bib11">11</xref>,<xref ref-type="bibr" rid="bib12">12</xref>,<xref ref-type="bibr" rid="bib13">13</xref>,<xref ref-type="bibr" rid="bib14">14</xref>,<xref ref-type="bibr" rid="bib15">15</xref></sup> consequently influencing the
overall RNAi efficiency. It was previously reported that chemically synthesized 29 base
duplex short hairpin RNAs that are substrates for Dicer are more potent RNAi triggers than
19 base duplex short hairpin RNAs.<sup><xref ref-type="bibr" rid="bib16">16</xref></sup> Similarly,
DsiRNAs of 25–30 nt can be up to 100-fold more potent than conventional 21-mer
duplexes targeted to the same sequence location.<sup><xref ref-type="bibr" rid="bib17">17</xref></sup> This increased potency might be attributed to the fact that
Dicer-generated 21–23-mer siRNAs are more efficiently incorporated into RISC through
the physical association of Dicer with the TAR RNA-binding protein and Argonaute
proteins.</p><p>Dicer cleavage of a blunt ended 27-mer duplex generally can generate a variety of
different 21–23-mers depending on its sequence parameters,<sup><xref ref-type="bibr" rid="bib15">15</xref>,<xref ref-type="bibr" rid="bib18">18</xref></sup> such as length/composition of
the 3′-terminus,<sup><xref ref-type="bibr" rid="bib19">19</xref></sup> GC content, inverted
repeats, etc. Furthermore, it has been demonstrated that siRNA efficacy is highly
dependent on the target position<sup><xref ref-type="bibr" rid="bib20">20</xref></sup> and RNAi
potency is susceptible to shifting a 21-mer siRNA even by a single base along the target
mRNA sequence.<sup><xref ref-type="bibr" rid="bib21">21</xref></sup> The overall RNAi efficacy of
DsiRNAs critically depends on the composition and potency of the processing products. Thus
acquiring a better understanding of DsiRNA designs is useful for enhancing RNAi efficacy.
In this study, we show that it is possible to predictably control dicing polarity and
strand selectivity <italic>via</italic> simply changing the sequences of the 3′-end
overhangs without altering the original duplex sequence. Recently, an asymmetric 25/27-mer
duplex having a 3′ two-nucleotide overhang and two 3′-DNA residues on the
blunt end has been rationally designed to obtain greater efficacy.<sup><xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib23">23</xref>,<xref ref-type="bibr" rid="bib24">24</xref></sup> This asymmetric structure directs dicing to predictably yield a
single primary cleavage product.</p><p>In the present study, a simple 5′-end labeled gel assay was performed to analyze
<italic>in vitro</italic> dicing patterns of chemically synthesized symmetric or asymmetric 25
base pair duplex <italic>TNPO3</italic> (<italic>Homo sapiens transportin 3</italic>) RNAs with different
3′ two-nucleotide overhangs. We observed that a series of heterogeneous products
were generated by Dicer cleavage from these substrates and Dicer preferentially processes
to a site 22 nt from the 3′-end of the substrates that have a two-base
ribonucleotide overhang. The 3′ two-base ribonucleotide overhangs can help orient
Dicer on the substrates and facilitate Dicer entry and cleavage. In contrast, the
direction and ability of human Dicer to generate siRNAs can be partially or completely
blocked by DNA residues at the 3′-termimi. Consistent with previous studies,
combining of such features into an asymmetric DsiRNA (25/27-mer DsiRNA) provides an
optimal design for obtaining a single, primary cleavage product with the best RNAi
potency.</p><p>To investigate the influence of the two-base 3′ overhang on dicing of substrates in
cells we employed Illumina Deep sequencing analyses to unravel the fates of the asymmetric
25/27-mer <italic>TNPO3</italic> duplexes in HEK293 cells. To validate the strand selection
process and target knockdown capabilities we also conducted dual-luciferase psiCHECK
reporter assays to monitor the RNAi potencies of both the “S” and
“antisense” (AS) strands derived from these DsiRNAs. In similarity to our
<italic>in vitro</italic> Dicer assays, the asymmetric duplexes were predictably processed into
the desired primary cleavage products of 21–22 nts in cells. We also observed
trimming of the 3′ ends and some additional Uracils added as well. Our observation
that the most efficacious strand was the most abundant revealed that the relative
frequencies of each “S” or “AS” strand are highly correlated with
the silencing activity and strand selectivity. A similar observation was also found in a
separate pair of asymmetric 25/27-mer duplexes targeting heterogeneous nuclear
ribonucleoprotein H (<italic>hnRNP H1</italic>), further validating the role of the sequence
composition of 3′ double-nucleotide overhang and the proposed optimal design
features.</p><p>Taken together, our data demonstrate that even though the only differences between a
family of DsiRNAs was the 3′ two-nucleotide overhang, dicing polarity and strand
selectivity are distinct depending upon the sequence and chemical nature of this overhang.
Thus, it is possible to predictably control dicing polarity and strand selectivity
<italic>via</italic> simply changing the 3′-end overhangs without altering the original
duplex sequence. These optimal design features of 3′-overhangs provide a facile
approach for rationally designing highly potent 27-mer DsiRNAs.</p></sec><sec sec-type="results"><title>Results</title><sec><title>Design of DsiRNAs against <italic>TNPO3</italic> and <italic>in vitro</italic> Dicing
approaches</title><p>We designed and synthesized a series of 25 base pair, two-base overhang containing RNAs
targeting the mRNA produced from the <italic>TNPO3</italic> gene (<italic>Homo sapiens transportin
3</italic>) which is one of the many HIV-1 dependency factors<sup><xref ref-type="bibr" rid="bib25">25</xref></sup> (<bold><xref rid="tbl1" ref-type="table">Table 1</xref></bold>). The original 21
(UU)/21 (UU) siRNA was previously reported to efficiently knockdown target gene
expression.<sup><xref ref-type="bibr" rid="bib25">25</xref></sup> The shifted 21-mer siRNAs
were also designed to target sites shifted downstream of the original 21-mer siRNA in
increments of 6 nt. Based upon the original 21-mer sequence, we designed the 27-mers
with an added six bases of the <italic>TNPO</italic> sequence The various arrangements of
3′ two-base overhangs are labeled as groups I and II, which include four symmetric
and six asymmetric DsiRNAs as listed in <bold><xref rid="tbl1" ref-type="table">Table 1</xref></bold>.</p><p>To evaluate the effects of the 3′-overhang in determining the position and
pattern of Dicer cleavage, P<sup>32</sup>-end labeled duplexes were processed <italic>in
vitro</italic> with recombinant human Dicer, and the dicing products were electrophoresed
in denaturing polyacrylamide gels and visualized by autoradiography. <bold><xref ref-type="fig" rid="fig1">Figure 1</xref></bold> depicts the two types of experimental approaches
used (method A and B). The dicing patterns were designated by the direction of Dicer
entry into the substrates. “L-R” indicates Dicer enters from left to right,
while the “R-L” model depicts Dicer processing from right to left. The
proportions of the cleaved/uncleaved fragments that result from “L-R” and
“R-L” dicing directions reflect the dicing efficiency for the 27-mer
duplex.</p></sec><sec><title>The 3′-overhangs influence <italic>in vitro</italic> dicing patterns</title><p>Initially, the products that result from <italic>in vitro</italic> digestion of group I
duplexes were analyzed using 5′-end P<sup>32</sup> labeled RNAs and a denaturing
gel electrophoresis assay. In method A, Dicer cleavage of the 5′-end
P<sup>32</sup> S strand-labeled duplexes generated two different sized S strand
fragments (<bold><xref ref-type="fig" rid="fig2">Figure 2a</xref></bold>). For “L-R” the
longer species 21–22 mer contained the P<sup>32</sup>, whereas for
“R-L” short 4–5 mers from the 25-mer S or 6–7 mers from the
27-mer AS strand, were produced, respectively. These results demonstrate that Dicer
enters these 27-mer duplex RNAs by either “L-R” or “R-L.” A
comparable proportion of long and short species (the ratio of “L-R” versus
“R-L” = 1) was observed in the 27 (UU)/27 (UU) duplex. However, the 27
(tt)/27 (UU) duplex with a two-base deoxyribonucleotide (tt) 3′ overhang on the S
strand yielded greater amounts of long “L-R” fragments and a very small
amount of “R-L” products (the ratio of “L-R” versus
“R-L” ≫1). In the reverse overhang setting 27 (UU)/27 (tt) duplex, the
ratio of “L-R” to “R-L” cleavage products was <1, confirming
that Dicer does not readily enter a duplex with dTdT 2-base overhangs. Furthermore, in
comparison with the 27 (tt)/27 (tt) duplex, the 27 (UU)/27 (tt) duplex has a dominant
“R-L” dicing polarity and the 27 (tt)/27 (UU) duplex has a major
“L-R” polarity, demonstrating that the 3′-UU overhang facilitates
Dicer binding and entry. A blunt end in the asymmetric duplexes (the 25 (GC)/27 (UU) and
the 25 (GC) /27 (tt) duplexes) seemed to show less dicing polarity.</p><p>To further validate these observations, we also 5′ P<sup>32</sup> end-labeled the
AS strand of the same 27-mer duplexes (method B) and preformed polyacrylamide denaturing
gel assays and autoradiography (<bold><xref ref-type="fig" rid="fig2">Figure 2b</xref></bold>). These
27-mer duplexes were also cleaved bidirectionally from both termini. The 27 (UU)/27 (UU)
duplex generated a comparable proportion of long and short species. In contrast, there
was very little processing of the 27 (tt)/27 (tt) duplex due to its two 3′-dTdT
overhangs. The 25 (GC)/27 (UU) and the 27 (tt)/27 (UU) duplexes containing a 3′-UU
overhang on the AS strand were readily processed from “L-R,” primarily
generating short fragments (<bold><xref ref-type="fig" rid="fig2">Figure 2b</xref></bold>), consistent
with the results observed when these duplexes were labeled on the S strand in which the
long fragments were the primary products (<bold><xref ref-type="fig" rid="fig2">Figure 2a</xref></bold>).
Only a trace amount of the “R-L” products were observed following dicing of
the above two duplexes due to the hindrance of the blunt end or 3′-dTdT overhang
on the S strand. As observed in <bold><xref ref-type="fig" rid="fig2">Figure 2a</xref></bold>, the dicing
pattern was completely reversed in the 27 (tt)/27 (UU) duplex and the 27 (UU)/27 (tt)
duplexes. These results definitively demonstrate that the 3′-overhang orients
Dicer entry and influences dicing preference, and DNA residues reduce the binding of
Dicer. Interestingly, a heterogeneous collection of 4–7-nt fragments was produced
by dicing of the asymmetric 27-mer duplexes (the 25 (GC)/27 UU and the 25 (GC)/27 (tt)).
These fragments may be derived from a second round of Dicer cleavage of these
siRNAs.</p></sec><sec><title>The 3′-overhangs affect RNAi potency</title><p>To investigate the influence of 3′-overhangs on RNAi activity, we measured the
target knockdown efficacy of the group I duplexes using a quantitative real-time reverse
transcription-PCR system (qRT-PCR) (<bold><xref ref-type="fig" rid="fig3">Figure 3</xref></bold>). The
original 21-mer siRNA was more potent than the shifted-21-mer (62 versus 40% knockdown
at 50 nmol/l and 40 versus 15% knockdown at 10 nmol/l). Compared with the
original 21-mer siRNA, the most potent knockdown was mediated by the 27 (tt)/27 (UU)
duplex, providing 78 and 60% knockdown at 50 and 10 nmol/l, respectively. The 25
(GC) /27 (UU) and 27 (UU)/27 (UU) duplexes enhanced (~10%) the RNAi potency, whereas
other 27-mer duplexes were slightly less potent than the original 21-mer siRNA. The
<italic>in vitro</italic> Dicer-cleavage reactions for the 27 (tt)/27 (UU) duplex
predominantly generated “L-R” cleavage products identical in size to the
original 21-mer siRNA. In contrast, major “R-L” dicing products were
produced from the 27 (UU)/27 (tt) duplex. The “L-R′ cleavage species
generate the more effective siRNAs.</p><p>The enhanced potency of the “L-R” cleavage might be attributed to
Dicer-generated products which result in preferential handoff of the AS strand to RISC.
Correspondingly, the unfavorable cleavage species deriving from “R-L” dicing
displayed reduced RNAi. These results suggest that the 27-mer duplex can yield a
specific, desired 21-mer species which is able to enhance RNAi activity, even though the
only differences among the group of 27-mer duplexes are the 3′-overhangs.</p></sec><sec><title>Asymmetric DsiRNAs containing a single 3′-DNA blunt terminus improve RNAi
potency</title><p>The “L-R” and “R-L” dicing patterns could be in equilibrium and
therefore compete with each other generating a mixture of siRNAs. By preventing one of
the two dicing directionalities <italic>via</italic> an unfavorable 3′-overhang it is
possible to promote a single Dicer entry pattern. Since the 3′-overhang plays a
determinant role in orienting Dicer entry and RNAi efficiency, it is possible to
generate the desired cleavage species <italic>via</italic> simply incorporating a favorable
3′ overhang and an unfavorable 3′ blunt end without altering the original
27-mer duplex sequence. For group I, 27-mer duplexes, the presence of a 3′-dTdT
overhang or an RNA containing blunt end did not completely abolish Dicer bidirectional
entry and different dicing patterns. To further restrict the dicing preference and entry
orientation, we designed a series of asymmetric duplexes (group II in <bold><xref rid="tbl1" ref-type="table">Table 1</xref></bold>) with a single two-nucleotide 3′-overhang on
the AS strand and two-nucleotide DNA residues at the 3′-blunt end of S strand, to
provide a single favorable Dicer entry and a restricted dicing pattern.</p><p><italic>In vitro</italic> Dicer processing of the group II duplexes was examined as previously
described (<bold><xref ref-type="fig" rid="fig4">Figure 4b,c</xref></bold>). As expected, the desired
“L-R” products derived from all these asymmetric duplexes were the
overwhelming majority indicating that the two-base deoxyribonucleotide 3′ blunt
end strongly impeded Dicer entry. When compared with the duplexes of group I, the
asymmetric group II duplexes greatly simplified the <italic>in vitro</italic> dicing pattern,
supporting the fact that terminal DNA residues impeded Dicer entry and can be used to
direct Dicer entry onto a DsiRNA to obtain a single desired cleavage product.</p><p>To further evaluate the asymmetric design, we evaluated the target knockdown efficacy
of these RNA duplexes (<bold><xref ref-type="fig" rid="fig4">Figure 4c</xref></bold>) <italic>via</italic> a
qRT-PCR assay. As we observed in the dicing reactions, the asymmetric duplexes with
various 3′ two-ribonucleotide overhangs had enhanced RNAi potency, whereas the
duplex having a 3′-dTdT overhang had less efficient target knockdown efficiency.
Moreover, the duplexes with a 3′ two-ribonucleotide overhang from group II showed
comparable knockdown efficacies (<bold><xref ref-type="fig" rid="fig4">Figure 4c</xref></bold>) as well
as dicing activities (<bold><xref ref-type="fig" rid="fig4">Figure 4b</xref></bold>).</p></sec><sec><title>The 3′-overhang sequences influence the guide strand selection of the
asymmetric DsiRNAs</title><p>To identify whether the sequence composition of the 3′ two-base overhang is
important for Dicer recognition and specificity, we used Illumina Deep sequencing
analyses to investigate the intracellular dicing products of the asymmetric 25/27-mer
duplexes transfected in HEK293 cells. At 48 hours post-transfection with
10 nmol/l of these DsiRNAs, total RNAs were isolated and prepared for Illumina
sequencing.</p><p>As shown in <bold><xref rid="tbl2" ref-type="table">Table 2a</xref></bold>, all the samples had similar
total reads. From the total reads specific sequences were collected and aligned using
the referenced sequences of the asymmetric DsiRNAs (AS or S strands). Surprisingly, the
data demonstrated that the total reads of these RNA duplexes and the proportion of the
AS strand to S strands were clearly distinct among the different duplexes (<bold><xref ref-type="fig" rid="fig5">Figure 5a,b</xref></bold>). Unequal transfection efficiencies or sample
processing might cause such differences; however, the relative strand distributions
suggest that the observed differences can largely be attributed to the 3′ overhang
compositions. The relative abundance of AS to S sequences declined relative to the
two-base overhangs as follows: GG > GC, AA > CC, UU ≫ tt. For example,
despite the close number of total reads for the 25 (gc)/27 (GG) duplex (477,396) and the
25 (gc)/27 (CC) duplex (431,423) these two duplexes have distinctly different strand
distributions with the relative ratios of AS to S being 7.06 and 1.21, respectively.
Similarly, the 25 (gc)/27 (UU) duplex (239,788) and the 25 (gc)/27 (tt) duplex (201,128)
have a similar number of total reads, whereas their relative strand distributions are
very different. Therefore, these data demonstrate that the sequence composition of the
3′ overhang plays an important role in determining the fates of these DsiRNAs with
respect to the dicing pattern, RISC loading and strand stability.</p><p>Manual inspection of the AS and S strands focusing only on the top 10 sequences,
narrowed the list to 60~70% of the total reads (<bold><xref rid="tbl2" ref-type="table">Table
2b</xref></bold>), but this analysis still maintained the same propensity for relative
abundance of AS/S (such as, GG > GC, AA > CC, UU ≫ tt). We further
identified the Dicer-cleaved RNA species for the top 10 sequences. <bold><xref ref-type="fig" rid="fig5">Figure 5c</xref></bold> and <bold>Supplementary Figure S1</bold> display two major dicing
patterns as previously determined: “L-R” cleavage generating the desired
siRNAs and “R-L” cleavage producing the undesired siRNAs. Consistent with
the <italic>in vitro</italic> dicing assay, the asymmetric duplexes having a 3′
two-ribonucleotide overhang were predictably processed into the desired
“L-R” cleavage products of 21-22 mers in cells (<bold>Supplementary Figure
S1</bold>). The population of “L-R” cleavage products is summarized in
<bold><xref rid="tbl2" ref-type="table">Table 2b</xref></bold>. Approximately 85% of the AS strands
were preferentially generated from “L-R” cleavage products. In contrast, the
AS strand of the 25 (gc)/27 (tt) duplex only produced 11.65% of the desired
“L-R” cleavage species, which corresponds with the <italic>in vitro</italic> dicing
results.</p><p>Interestingly, we also observed RNA editing of both strands, such as trimming of a
single or double-nucleotide on the 3′ end or addition of untemplated nucleotides
to the 3′ or 5′ termini. As shown in <bold><xref ref-type="fig" rid="fig5">Figure
5d</xref></bold> and <bold>Supplementary Figure S1</bold>, trimming of the 3′ end
occurred on both strands, especially when DNA residues were incorporated into the
overhangs or the blunt end. Untemplated nucleotides were added to the 3′ or
5′-termini, most likely following the trimming or dicing reactions. For example,
the 3′-GC overhang on the AS strand of the 25 (gc)/27 (GC) duplex was trimmed by a
single “C” and subsequently extended by a U or A (<bold>Supplementary Figure
S1a</bold>, entry 7 and 8). For the 25 (gc)/27 (tt) duplex, after its 3′ DNA (gc)
blunt end on the S strand was trimmed, the resulting RNA was subjected to uridylation of
the 3′ end (<bold>Supplementary Figure S1b</bold> S, entry 6, 7, and 10). Moreover, we
also found that nucleotides most frequently added to these duplexes were U and A. The
extension of a U or A was much more prevalent on the 3′ end than on the 5′
end. Although the RNA editing appeared to be widespread among all the duplexes, the
frequency is generally lower than 10% of the total reads. The highest frequency of
editing exceeded 50% in the 25 (gc)/27 (GC) duplex, suggesting the 3′ overhang
composition also affects RNA extension and ultimately influences the Dicer-cleavage site
and RNAi potency.</p></sec><sec><title>The 3′-overhang sequence compositions influence strand selectivity and RNAi
activity of asymmetric DsiRNAs</title><p>It is known that selection of the guide strand depends upon the differences in the
thermodynamic stability of the two ends of the 21/22-mer duplexes.<sup><xref ref-type="bibr" rid="bib5">5</xref>,<xref ref-type="bibr" rid="bib26">26</xref></sup> The 3′
two-nucleotide overhangs can alter the stability of the duplex ends, eventually
affecting strand selection by the Argonaute proteins. Argonaute 2 binds the guide strand
in an oriented manner which facilitates cleavage of the passenger strand of the siRNA
during RISC loading.<sup><xref ref-type="bibr" rid="bib27">27</xref></sup> The potential for
competition of the passenger strand for RISC entry necessitates proper design strategies
to optimize the desired guide strand selection and to minimize off target activity by
the undesired passenger strand.</p><p>To evaluate the RNAi activities of the asymmetric duplexes, we used the psiCHECK
reporter system in which the target sequence for the AS or S strands are inserted in the
3′ UTR of the <italic>Renilla luciferase</italic> gene. The siRNA-mediated inhibition of
luciferase activity for both the S and AS target orientations were independently tested
(<bold><xref ref-type="fig" rid="fig6">Figure 6a</xref></bold> and <bold><xref rid="tbl2" ref-type="table">Table
2c</xref></bold>). The strand selectivity was calculated as a measure of the relative
target inhibition efficiencies for each target orientation (<bold><xref ref-type="fig" rid="fig6">Figure
6b</xref></bold> and <bold><xref rid="tbl2" ref-type="table">Table 2c</xref></bold>). When compared with
the duplex harboring a 3′ dTdT overhang, all the duplexes with 3′ two
base-ribonucleotide overhangs tested in this study showed superior inhibition of
luciferase expression (IC<sub>50</sub> 20–100 pmol/l) when using the AS
guide strand against the S target. However, when the S strands were used as guides for
the AS orientation of the target, relatively lower efficiencies of target inhibition
were observed (IC<sub>50</sub> 200–8,000 pmol/l). The strand selectivity
(<bold><xref ref-type="fig" rid="fig6">Figure 6b</xref></bold>) favoring the AS strand relative to
the S strand as guides for these asymmetric duplexes demonstrated that the asymmetric
design favors handoff to RISC of the AS strand as guide following dicing. Moreover,
there was a profound bias in AS versus S strand function for the 3′ GG overhang.
The data revealed that the most efficacious strand was the most abundant and the
relative frequencies of the “S” or “AS” strands are highly
correlated with the silencing activity and strand selectivity. In brief, the rankings
for the RNAi efficiency for of the various 3′ overhangs is GG > GC, CC, AA >
UU ≫ tt. Correspondingly, the abundance of the “L-R” products for the
various 3′ two-base overhangs is also GG > GC, CC, AA > UU ≫ tt.</p></sec><sec><title>Validation of the role of the two-base 3′-overhang for a different pair of
asymmetric DsiRNAs</title><p>To verify the role of the 3′-overhang in guide strand selection and function, we
analyzed a separate set of asymmetric DsiRNAs that target <italic>hnRNP H1</italic>. These two
asymmetric DsiRNAs differ by a single nucleotide, but have strikingly different
functional activities, and thus represent an interesting test for the role of the
two-base 3′ overhangs (<bold><xref rid="tbl3" ref-type="table">Table 3a</xref></bold>). The RNAi
potencies of these RNAs were also evaluated using the psiCHECK system. As depicted in
<bold><xref rid="tbl3" ref-type="table">Table 3b</xref></bold>, the knockdown efficiencies of both the
S and AS strands of these asymmetric <italic>hnRNP H1</italic> DsiRNAs were independently
assessed and the strand selectivity was calculated.</p><p>There is only a one-base shift along the target mRNA sequence between the site 325
DsiRNAs and the site 324 DsiRNAs. The site 324 DsiRNAs showed better overall RNAi
efficiency (combined knockdown of both S and AS targets) when compared with the site 325
DsiRNAs containing the same 3′-overhangs. The site 324 DsiRNAs showed a comparable
knockdown efficiency for either the S or AS strands as guides against their
corresponding targets (<bold><xref rid="tbl3" ref-type="table">Table 3b</xref></bold>). Despite the weak
strand selectivity for the 324 DsiRNAs, there was a relative tendency for biased strand
selectivity for the 25 (gg)/27 (GG) duplexes compared to the 3′-CC, UU and AA
overhangs. Substantial strand selectivity was observed for the site 325 DsiRNAs. For
example, the selectivity of AS to S of the 25 (tg)/27 (GG) duplex is 6.73 and the 25
(tg)/27 (AA) duplex is 8.86, respectively. For the site 325 DsiRNAs it was interesting
to see that the 3′-AA overhang provided the best RNAi activity and strongest
strand selectivity. In this case, the 3′-AA overhang makes the siRNA completely
complementary to the <italic>hnRNP H1</italic> target site, implying a perfectly matched
3′-overhang probably contributes to enhanced RNAi activity. Additionally, we also
chose two representative DsiRNAs from each site to evaluate the target knockdown
efficacy <italic>via</italic> a qRT-PCR assay (<bold>Supplementary Figure S3</bold>). Consistent with
our observation in the <italic>TNPO3</italic> case, the asymmetric duplexes with perfectly
matched 3′-overhangs have enhanced RNAi potency, whereas the duplex having a
3′-dTdT overhang had less efficient target knockdown efficiency. For example, the
site 325 DsiRNA 25 (tg)/27 (AA) showed a lower IC<sub>50</sub> value
(44.37 pmol/l) compared with the 25 (tg)/27 (tt) duplex (IC<sub>50</sub> =
284 pmol/l).</p><p>We also used Illumina Deep sequencing analyses to interrogate the relative strand
abundance of the <italic>hnRNP H1</italic> asymmetric 25/27-mer duplexes. Total RNAs were
isolated and prepared for Illumina Deep sequencing at 24 hours post-transfection of
these DsiRNAs in HCT116 cells. According to their RNAi potency and strand selectivity,
we chose two representative DsiRNAs for each target site. For example, the site 324
DsiRNAs 25 (tg)/27 (GG) and 25 (tg)/27 (AA) that showed a big difference in RNAi
activity and selectivity were selected for deep sequence analyses. Similarly, deep
sequencing analyses were performed with the DsiRNA 325 25 (gg)/27 (GG) and 25 (gg)/27
(CC) duplexes. To simplify the comparisons we only focused on the top 10 sequences. As
shown in <bold><xref rid="tbl3" ref-type="table">Table 3b</xref></bold> and <bold><xref ref-type="fig" rid="fig7">Figure
7a,b</xref></bold>, the total reads of these RNA duplexes and the proportions of the
AS to S strands were distinct among the experimental duplexes. In similarity to our
previous observation with the <italic>TNPO3</italic> DsiRNAs, the relative abundances of the AS
to S strands for both site 324 and 325 DsiRNAs were consistent with the strand
selectivity in the RNAi assays. For example, the relative distribution of AS to S
(<bold><xref ref-type="fig" rid="fig7">Figure 7b</xref></bold>) ratios ranked the 25 (tg)/27 (GG)
≫ 25 (tg)/ 27 (AA) for site 324, and GG > CC for site 325 which is the same
trend for strand selectivity shown in <bold><xref rid="tbl3" ref-type="table">Table 3b</xref></bold>. Thus
these observations validated that the 3′-overhang composition contributes to the
fates of the DsiRNAs and ultimately RNAi activity.</p><p>As previously described for the <italic>TNPO3</italic> DsiRNA system, the two major
Dicer-cleavage products (“L-R” and “R-L”) in the top 10
sequences were identified (<bold><xref ref-type="fig" rid="fig7">Figure 7c</xref></bold> and
<bold>Supplementary Figure S2</bold>). <bold><xref rid="tbl3" ref-type="table">Table 3c</xref></bold> lists the
population of “L-R” cleavage products producing the desired siRNA species.
Consistent with the results obtained with the <italic>TNPO3</italic> system, >80% of the AS
strands or S strands of the <italic>hnRNP H1</italic> DsiRNAs preferentially produced the
desired primary “L-R” cleavage products. As shown in <bold>Supplementary Figure
S2</bold>, RNA editing took place at the ends of both strands as previously observed. For
example, trimming of a single or double-nucleotide at the 3′ end occurred often on
the AS strand. Also 1 or 2 untemplated nucleotides (such as “U,”
“C,” “CC,” “UU,” or “CU”) were most
frequently added to the 3′ end of both strands. Although RNA editing appears to be
a widespread phenomenon among all the tested duplexes, the mechanism is unknown.</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>The RNAse III family member Dicer initiates RNAi by processing double-stranded RNAs into
21–23 nt double-stranded RNAs (either miRNAs or siRNAs) generating a two-base
3′-overhang.<sup><xref ref-type="bibr" rid="bib7">7</xref>,<xref ref-type="bibr" rid="bib28">28</xref></sup> In association with Dicer, the siRNA products are loaded into RISC
such that only one of the original strands is incorporated and used as a guide for the
sequence-specific post-transcriptional silencing of cognate genes. The remaining strand,
known as the antiguide or passenger strand, is degraded. Previous studies have
demonstrated in mammals that the PAZ domain of Dicer is a single-stranded RNA-binding
module that has preference for two-base single strand overhangs produced by another RNAse
III family member Drosha, which processes primary miRNAs into pre-miRNAs.<sup><xref ref-type="bibr" rid="bib6">6</xref>,<xref ref-type="bibr" rid="bib11">11</xref>,<xref ref-type="bibr" rid="bib29">29</xref></sup> The PAZ/PIWI domains of the Ago2 protein serve as anchors to
spatially orient the bound RNA substrates in the enzyme active site.<sup><xref ref-type="bibr" rid="bib30">30</xref>,<xref ref-type="bibr" rid="bib31">31</xref></sup> Therefore, DsiRNAs
with two-nucleotide 3′-overhangs that are favorable for Dicer binding/cleavage and
subsequent Ago anchoring are believed to enhance RNAi potency.<sup><xref ref-type="bibr" rid="bib9">9</xref></sup> Addition of RNA tetraloops, unfavorable DNA residues or fluorescent
groups at the ends of double-stranded RNAs have been demonstrated to partially or
completely block cleavage by human Dicer.<sup><xref ref-type="bibr" rid="bib6">6</xref>,<xref ref-type="bibr" rid="bib16">16</xref></sup></p><p>The single-stranded two-base 3′ overhangs present in traditional 21-mer siRNAs are
required for optimal siRNA function. As a regular practice in designing siRNAs, the
two-base 3′-deoxynucleotide overhangs (such as “tt”) are often added to
19-mer siRNAs, often without regard to complementarity with the target sequence. However,
here we find that when this feature is applied to 25/27-mer DsiRNAs, it adversely affects
dicing and subsequent RNAi activity. Moreover, we also find that the sequence composition
of ribose 3′ two-base overhangs significantly affect dicing polarity and strand
selectivity.</p><p>We first carried out investigations of <italic>in vitro</italic> dicing products using symmetric
25 base pair duplexes with various overhang configurations (group I, <bold><xref rid="tbl1" ref-type="table">Table 1</xref></bold>). Our results showed that a 3′ tt overhang attenuates
Dicer entry onto the substrate while 3′ UU overhangs are favorable for Dicing entry
and subsequent cleavage. When Dicer enters the substrate from either end of a duplex
(“L-R” or “R-L” models), resulting in heterogeneous cleavage
products, these can consequently impact on RNAi potency. Even though the only minor
difference among all the symmetric 27-mer DsiRNAs tested is the sequence composition of
the two-base 3′ overhang, the 27 (tt)/27 (UU) DsiRNA preferentially generated more
“L-R” dicing products generating the desired siRNAs with the highest target
knockdown efficiencies, whereas other duplexes with a 3′ tt overhang on the AS
strand were primarily processed by “R-L” Dicer entry generating siRNAs with
poor efficacy. Because a 3′=tt overhang or a ribose blunt end does not completely
abolish Dicer entry/cleavage in the group I design, bidirectional dicing products were
still observed. We further restricted the dicing preference in order to obtain the desired
“L-R” product through rational design of 3′-termini. These optimized
asymmetric DsiRNAs (group II, <bold><xref rid="tbl1" ref-type="table">Table 1</xref></bold>) have a single,
favorable 3′ two-ribonucleotide overhang on the AS strand and an unfavorable
two-nucleotide blunt DNA residue on the 3′ end of the S strand<sup><xref ref-type="bibr" rid="bib22">22</xref></sup> which simplifies the Dicing pattern, predictably
generating a single, desired “L-R” product, thereby enhancing RNAi potency. It
is noteworthy that these design features can provide an optimal structure for binding by
the Dicer PAZ domain along with an anchor site for Ago2 in RISC. The increased RNAi
efficacy mediated by the 27-mer DsiRNAs can be attributed to two features (i) the major
“L-R” products generate siRNAs with desired sequences; and (ii) the
“L-R” products that have a 3′ two-base overhang on the AS strand result
in preferential utilization of the AS strand as a guide in the RNAi machinery.</p><p>Since “L-R” and “R-L” Dicer entry patterns can coexist in a
dicing reaction, they will compete each other. By blocking “R-L” dicing
<italic>via</italic> the 3′-DNA blunt end on the S strand and simultaneously facilitating
“L-R” entry <italic>via</italic> optimized 3′ two-base ribonuleotide overhangs
on the desired AS/guide strand, it is possible to significantly promote the desired
“L-R” dicing products and strand selectivity as previously
demonstrated.<sup><xref ref-type="bibr" rid="bib22">22</xref></sup> Furthermore, the RNA
slicing-based silencing pathway is involved in multiple cycles of target binding, cleavage
and product release mediated by the Argonaute 2 protein. In this scenario, the Argonaute 2
protein remains bound to the guide strand promoting guide strand selection by slicing the
passenger strand, thereby establishing and protecting the guide strand from
degradation.<sup><xref ref-type="bibr" rid="bib4">4</xref>,<xref ref-type="bibr" rid="bib5">5</xref></sup></p><p>We have been interested in determining the influence of the sequence composition of the
two-base 3′ overhangs on Dicer processing and strand selectivity. We have examined
the fates of asymmetric <italic>TNPO3</italic> duplexes in cells by Illumina Deep sequencing
analyses. Additionally the RNAi potencies of both the “S” and “AS”
strands derived from these duplexes were also evaluated by psiCHECK reporter gene assays.
In similarity to our <italic>in vitro</italic> Dicer assays, the asymmetric duplexes with two-base
3′ ribonucleotide overhangs were predictably and primarily processed into the
desired “L-R” cleavage products of 21–22 mers in cells (<bold><xref rid="tbl2" ref-type="table">Table 2</xref></bold> and <bold>Supplementary Figure S1</bold>).</p><p>The effect of the sequence composition of the two-base 3′ overhang is an important
point of discussion. In several previous studies, a preference in Dicer binding and
Dicer-cleavage efficiency for DsiRNAs containing purine/purine nucleotide 3′
overhangs over pyrimidine/pyrimidine 3′-overhangs was observed. Dicer binding and
activity was ordered by the 3′ overhangs CC > GC > GG > AA >
UU,<sup><xref ref-type="bibr" rid="bib15">15</xref></sup> GG > AA > UU > CC >
tt,<sup><xref ref-type="bibr" rid="bib9">9</xref></sup> and AA > GC ≫ GG > CC >
UU.<sup><xref ref-type="bibr" rid="bib19">19</xref></sup> However, other studies showed
different tendencies. For example, a molecular dynamic simulation study<sup><xref ref-type="bibr" rid="bib32">32</xref></sup> indicated that the 3′-UU overhang made a
relatively more stable complex with the PAZ domain compared to 3′-GG, AA, and CC
overhangs (UU > GG > AA > CC). In addition, an asymmetric 27 mer DsiRNA with a
3′-UU overhang on the AS strand was shown to be the most potent inhibitor of gene
expression compared to DsiRNAs having different sequence compositions of the 3′
overhang (UU > GG, GC, CC > AA).<sup><xref ref-type="bibr" rid="bib17">17</xref>,<xref ref-type="bibr" rid="bib22">22</xref></sup> It was noted that in this case the 3′-UU overhang
was completely complementary to the target, implying a perfectly matched 3′-overhang
may contribute to RNAi activity. Our data for both the <italic>TNPO3</italic> and <italic>hnRNP
H1</italic> targets also demonstrated that asymmetric DsiRNAs with a 3′-overhang
complementary to the target mRNA have enhanced RNAi potency. Although the optimal sequence
composition of the 3′ termini is still controversial overall for RNAi, the 3′
overhang composition undoubtedly contributes to Dicer binding and RNAi potency. Our data
further demonstrate that the composition of the 3′ two-base overhangs significantly
influences the relative strand abundance and selectivity into RISC. Our observation that
the most efficacious strands were also the most abundant revealed that the relative
frequencies of “S” or “AS” strands are highly correlated with
overall strand selectivity and hence silencing activity. For example, in the
<italic>TNPO3</italic> DsiRNA system, there is a strong bias in the abundance of
“L-R” products and selection of the AS strand with the order of preference for
two-base 3′ overhang being GG > GC, CC, AA > UU ≫ tt. Similarly, the
strand distribution (Ratio of AS to S) follows the same 3′ overhang order of GG >
GC, AA > CC, UU ≫ tt. Similar observations on biases dictated by the two-base
overhang were also found for another set of asymmetric 25/27-mer duplexes targeting the
<italic>hnRNP H1</italic> mRNA further validating the significance of the role of the sequence
composition of the 3′ two-base overhang. With both the <italic>TNPO3</italic> and <italic>hnRNP
H1</italic> mRNAs, our results support the observation that DsiRNAs with purine/purine (GG,
AA) nucleotide overhangs are generally preferred over pyrimidine/pyrimidine overhangs (CC,
UU).</p><p>In addition to the role of the two-base overhang in DsiRNA selection and RNAi, we have
observed two types of RNA editing on both strands of the processed DsiRNAs. These are
trimming of a single or double-nucleotide on the 3′ end and addition of untemplated
nucleotides to the 3′ or 5′ termini subsequent to the trimming (<bold><xref ref-type="fig" rid="fig5">Figure 5d</xref></bold> and <bold>Supplementary Figures S1</bold> and <bold>S2</bold>).
The deep sequences reveal that the trimming of the 3′ end occurs on both strands
following Dicer processing. There is more trimming when DNA residues were incorporated
into the overhangs or blunt end. Moreover, the nucleotides most frequently added to these
duplexes are U and A. The addition of a U or A was much more prevalent on the 3′ end
than on the 5′ end. Interestingly, the highest frequency of RNA editing, which
exceeded 50% of the sequences analyzed was observed for the <italic>TNPO3</italic> 25 (gc)/27 (GC)
duplex, in which the 3′ end of the AS was extended by a U or A. Considering the
direct relationship between sequence abundance and RNAi activity of the AS strand, we
conjecture that the untemplated nucleotide additions to the 3′ end of the AS strand
somehow facilitate RISC loading. It remains to be investigated what enzymes are
participating in the RNA editing. We do not know if the addition of untemplated
nucleotides takes place before or after Dicer cleavage.</p><p>In conclusion, consistent with previous reports,<sup><xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib24">24</xref></sup> our data demonstrate that
3′ RNA residues are more favorable than DNA residues for Dicer processing of
DsiRNAs. Dicer entry onto DsiRNAs is oriented by the nature of the 3′ ends. In
addition, the entry of Dicer also influences guide strand selection and ultimately RNAi
potency. Since the PAZ domain is sensitive to the type of 3′ overhang sequence
composition,<sup><xref ref-type="bibr" rid="bib6">6</xref></sup> Dicing patterns could be
predictably controlled through the rational design of the 3′ end. Furthermore, the
sequence composition of the 3′ two-base overhang appears to influence RNA editing of
the siRNAs, as well as the Dicing pattern and recruitment of the siRNA guide strand into
the RISC. Further research will be needed to understand the mechanism of RNA recognition
and editing events in the RNAi pathway.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><p><italic>Materials</italic>. Unless otherwise noted, all chemicals were purchased from
Sigma-Aldrich (St Louis, MO), T4 PNK enzymes and buffer were obtained from New England
BioLabs (Ipswich, MA) and all cell culture products were purchased from GIBOC (Gibco
BRL/Life Technologies (Carlsbad, CA), a division of Invitrogen, Carlsbad, CA). The cell
lines HEK 293 and HCT116 are from the ATCC (Manassas, VA). Random primers (Invitrogen);
Lipofectamine 2000 (Invitrogen).</p><p><italic>siRNAs</italic>. All the siRNAs were synthesized and purified using high-performance
liquid chromatography at Integrated DNA Technologies (Coralville, IA). All RNA duplexes
against <italic>TNPO3</italic> (group I and group II) used in this study are listed in the
<bold><xref rid="tbl1" ref-type="table">Table 1</xref></bold>. All RNA duplexes against <italic>hnRNP H1</italic>used in this study are listed in the <bold><xref rid="tbl3" ref-type="table">Table 3a</xref></bold>.</p><p><italic>In vitro Dicer assays</italic>. All the S strands and AS strands were end-labeled with T4
polynucleotide kinase and े-<sup>32</sup>P-ATP. Unlabeled S or AS RNAs were
annealed with equal molar amounts of 5′-end-labeled corresponding AS or S strands in
HBS buffer in order to form siRNA duplexes. siRNA duplexes (1 pmol) were incubated
at 37 °C for 40 minutes in the presence or in the absence of 1 U of
human recombinant Dicer enzyme following the manufacturer's recommendations (Ambion,
Austin, TX). Reactions were stopped by phenol/chloroform extraction and the resulting
solutions were electrophoresed in a 20% polyacrylamide denaturing gel. The gels were
subsequently exposed to X-ray film.</p><p><italic>Cell culture</italic>. HEK 293 cells and HCT116 cells were purchased from ATCC and
cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum according to their respective data sheets. Cells were cultured in a
humidified 5% CO<sub>2</sub> incubator at 37 °C.</p><p><italic>Determination of TNPO3 gene silencing (qRT-PCR analysis)</italic>. HEK 293 cells were
split in 24-well plates to 60–70% confluency in Dulbecco's modified
Eagle's medium media 1 day before transfection. The cells were transfected with 10
or 50 nmol/l of experimental anti-<italic>TNPO3</italic> duplex RNAs using Lipofectamine
2000 following the manufacturer's recommendations (Invitrogen). Forty eight hours
post-transfection total RNAs were isolated with TriZol reagent (Invitrogen). Expression of
the <italic>TNPO3</italic> gene was analyzed by quantitative real time-PCR using a 2x iQ
SyberGreen Mastermix (Bio-Rad) and specific primer sets at a final concentration of
400 nmol/l. Primers were as follows: <italic>TNPO3</italic> forward primer: 5′-CCT
GGA AGG GAT GTG TGC-3′ <italic>TNPO3</italic> reverse primer: 5′-AAA AAG GCA AAG AAG
TCA CAT CA-3′ GAPDH forward primer: 5′ -CAT TGA CCT CAA CTA CAT G-3′
GAPDH reverse primer: 5′-TCT CCA TGG TGG TGA AGA C-3′.</p><p>TriZol reagent was used to extract total RNA according to the manufacturer's
instruction (Invitrogen). Residual DNA was digested using the DNA-free kit per the
manufacturer's instructions (Ambion). cDNA was produced using 2 µg of total
RNA, Moloney murine leukemia virus reverse transcriptase and random primers in a 15
µl reaction according to the manufacturer's instructions (Invitrogen).GAPDH
expression was used for normalization of the qPCR data.</p><p><italic>Illumina Deep sequence and data analysis</italic>. HEK 293 cells were split into 24-well
plates at 60–70% confluency in Dulbecco's modified Eagle's medium media
one day prior to transfection. The cells were transfected with 10 nmol/l of the
asymmetric group II RNA duplexes using Lipofectamine 2000 following the
manufacturer's recommendations (Invitrogen). Forty eight hours post-transfection the
total RNAs were isolated with TriZol reagent (Invitrogen) and prepared for Illumina Deep
sequencing.</p><p>To identify the most frequent S and antisense products from each of the DsiRNA molecules,
the sequences generated from Illumina Pipeline v1.6 were aligned with the S and AS strands
of each siRNA molecule using Novoalign v2.05 (<ext-link ext-link-type="uri" xlink:href="http://www.novocraft.com">http://www.novocraft.com</ext-link>). All subsequent analyses were carried out
using the R statistical environment and Bioconductor packages “Biostrings” and
“ShortRead.”<sup><xref ref-type="bibr" rid="bib33">33</xref></sup> Only sequences
that could be aligned to the siRNA sequences without mismatches were retained. The
relative starting and ending positions of the siRNA sequences were determined based on
their aligned positions and lengths, and the frequency of each product was counted. Only
the 10 most frequent products are reported.</p><p>To examine whether there are nucleotide additions or deletions at either end of the Dicer
processed products, the raw sequences were matched to the siRNA AS sequence with a seed
size of 16 after removing the 3′-adapter using the Bioconductor package
“ShortRead.” For example, for a siRNA sequence length of 23, the Illumina
sequences were aligned totally to eight seeds which are the subsequences from bases
1–16, 2–17, and so on, of the original siRNA sequence. The matched sequences
were then reduced to a set of unique sequences along with their number of occurrences.
This set of sequences was then aligned with the siRNA reference sequence using the
ClustalX2 multiple alignment tool<sup><xref ref-type="bibr" rid="bib34">34</xref></sup> not allowing
gaps. The multiple aligned sequences were visualized and exported using
JalView.<sup><xref ref-type="bibr" rid="bib35">35</xref></sup> The extra bases at either end of
the product were highlighted manually.</p><p><italic>Dual luciferase assay (detection of IC<sub>50</sub> value</italic>). The 45 base pair
oligomer of <italic>TNPO3</italic> cDNA was inserted into the <italic>Spe</italic>I and <italic>Xho</italic>I
restriction endonuclease sites downstream of the humanized <italic>Renilla luciferase</italic>gene in the psiCHECK-2 vector (Promega, Fitchburg, WI) to generate plasmids
psiCHECK-<italic>TNPO3</italic>-AS (passenger strand reporter) and psiCHECK-<italic>TNPO3</italic>-S
(guide strand reporter). HCT116 cells were cotransfected in a 96-well format (25,000
cells/well) with 10 ng of the respective psiCHECK-<italic>TNPO3</italic>-AS or
psiCHECK-<italic>TNPO3</italic>-S vector, 100 fmol/l–50 nmol/l DsiRNAs and
0.1 µl Lipofectamine2000 (Invitrogen) per well. Cells were lysed in 1× Passive
Lysis Buffer (Promega) 24 hours after transfection and analyzed using the Dual-Luciferase
Reporter System (Promega) on a Veritas microplate luminometer (Turner Biosystems,
Sunnyvale, CA). The average values were calculated from three replicates to set
<italic>Renilla/Firefly</italic> luciferase expression to 100%. An IC<sub>50</sub> curve was
generated using Prism 5.01 software (GraphPad, La Jolla, CA). Sigmoidal dose responses
were calculated according to <italic>Y</italic> =Bottom + (Top − Bottom)/(1 + 10)<italic>Y</italic>((LogEC<sub>50</sub> – <italic>X</italic>)); where <italic>X</italic> is the logarithm of
concentration and <italic>Y</italic> is the response.</p><p><italic>The sequences of Oligomers</italic>.</p><p><italic>TNPO3</italic> AS</p><p><italic>TNPO3</italic> AS_S: 5′-CCg CTCGAG ggagcaaagc cgacattgca gctcgtgtac caggcagtgc
aggcg ACTAGT CC-3′</p><p><italic>TNPO3</italic> AS_AS: 5′-GG ACTAGT cgcct gcactgcctg gtacacgagc tgCaatgtcg
gctttgctcc CTCGAG CGG-3′</p><p><italic>TNPO3</italic> sense(S)</p><p><italic>TNPO3</italic> S_S: 5′-CCG CTCGAG cgcct gcactgcctg gtacacgagc tgCaatgtcg gctttgctcc
ACTAGT CC-3′</p><p><italic>TNPO3</italic> S_AS: 5′-GG ACTAGT ggagcaaagc cgacattgca gctcgtgtac caggcagtgc aggcg
CTCGAG CGG-3′</p><p>The fragment of 343 base pair in <italic>hnRNP H1</italic> cDNA includes the region of bases
90–432 in the reference sequence NM_005520. The reporter plasmids psi-<italic>hnRNP
H</italic>-S (sense reporter) and psi-<italic>hnRNP H</italic>-AS (AS reporter) were derived by
cloning the hnRNPH sequences in the 3′-UTR of the humanized <italic>Renilla
luciferase</italic> gene in the psiCHECK-2 (Promega).<sup><xref ref-type="bibr" rid="bib22">22</xref></sup></p><p><xref ref-type="supplementary-material" rid="sup1"><bold>SUPPLEMENTARY MATERIAL</bold></xref>
<bold>Figure S1.</bold> Illumina Deep sequence analysis of asymmetric <italic>TNPO3</italic> 27-mer
Dicer-substrate siRNAs (group II).
<bold>Figure S2.</bold> Illumina Deep sequence analyses of asymmetric 27-mer <italic>hnRNP H1</italic>Dicer-substrate siRNAs.
<bold>Figure S3.</bold> Silencing of <italic>hnRNP H1</italic> by the site 324 and 325 duplex
DsiRNAs.</p></sec></body><back><ack><p>We thank Kumi Sakurai, Britta Hoehn, and Soifer Harris for helpful discussions. We thank
City of Hope DNA sequencing core (Harry Gao and Jinhui Wang) for Illumina Deep Sequencing
and City of Hope Bioinformatics Core facility (Xiwei Wu and Haiqing Li) for data analyses.
This work was supported by grants from the National Institutes of Health awarded to J.J.R.
AI29329, AI42552, and HL07470. J.J.R. and M.A.B. are cofounders of Dicerna Pharmaceuticals,
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workbench</article-title><source>Bioinformatics</source><volume>25</volume><fpage>1189</fpage><lpage>1191</lpage><pub-id pub-id-type="pmid">19151095</pub-id></mixed-citation></ref></ref-list><sec sec-type="supplementary-material" id="sup1"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="xob1"><label>Figure S1.</label><caption><p>Illumina Deep sequence analysis of asymmetric <italic>TNPO3</italic> 27-mer Dicer-substrate
siRNAs (group II).</p></caption><media mimetype="application" mime-subtype="pdf" xlink:href="mtna20126x1.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="xob2"><label>Figure S2.</label><caption><p>Illumina Deep sequence analyses of asymmetric 27-mer <italic>hnRNP H1</italic> Dicer-substrate
siRNAs.</p></caption><media mimetype="application" mime-subtype="pdf" xlink:href="mtna20126x2.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="xob3"><label>Figure S3.</label><caption><p>Silencing of <italic>hnRNP H1</italic> by the site 324 and 325 duplex DsiRNAs.</p></caption><media mimetype="application" mime-subtype="pdf" xlink:href="mtna20126x3.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></back><floats-group><fig id="fig1"><label>Figure 1</label><caption><p><bold><italic>In vitro</italic> Dicer processing of (a) 5</bold>′
<bold>P</bold><sup><bold>32</bold></sup><bold>-end labeled sense or antisense (b) duplexes.</bold> Two
types of experimental approaches are displayed as method A and B, respectively. The
dicing patterns are named by the direction of Dicer entering the duplex as left to right
(L-R) and right to left (R-L).</p></caption><graphic xlink:href="mtna20126f1"></graphic></fig><fig id="fig2"><label>Figure 2</label><caption><p><bold><italic>In vitro</italic> Dicer cleavage of 5</bold>′ <bold>P</bold><sup><bold>32</bold></sup><bold>-end
labeled sense or antisense strands.</bold> Group I duplexes. The cleavage fragments that
result from dicing L-R and R-L were visualized autoradiographically following denaturing
gel electrophoresis of the Dicer-cleavage products: (a) sense and (b) antisense. The two
experimental approaches (method A and B) are defined in the legend to Figure 1.</p></caption><graphic xlink:href="mtna20126f2"></graphic></fig><fig id="fig3"><label>Figure 3</label><caption><p><bold>Silencing of <italic>Homo sapiens</italic> transportin 3 (<italic>TNPO3</italic>) by group I duplex
Dicer-substrate small interfering RNAs (DsiRNAs)</bold>. HEK 293 cells were transfected
with 50 or 10 nmol/l of the experimental anti-<italic>TNPO3</italic> duplex DsiRNAs.
<italic>TNPO3</italic> mRNA levels were detected by quantitative real-time reverse
transcription (qRT-PCR). The data are normalized with GAPDH mRNA levels and represent
the average of three replicate assays.</p></caption><graphic xlink:href="mtna20126f3"></graphic></fig><fig id="fig4"><label>Figure 4</label><caption><p><bold><italic>In vitro</italic> Dicer cleavage of 5</bold>′ <bold>P</bold><sup><bold>32</bold></sup><bold>-end
labeled sense or antisense strands</bold>. Group II asymmetric duplex Dicer-substrate
small interfering RNAs (DsiRNAs) and RNA interference (RNAi) potency. The cleavage
fragments that result from dicing L-R and R-L were visualized by autoradiography of the
P<sup>32</sup> 5′ end-labeled strands: (<bold>a</bold>) sense and (<bold>b</bold>)
antisense. Two experimental approaches (method A and B) and the dicing pattern are
defined the legend to Figure 1. (<bold>c</bold>) Silencing of <italic>Homo sapiens</italic>transportin 3 (<italic>TNPO3</italic>) triggered by group II duplex RNAs. HEK 293 cells were
transfected with 50 or 10 nmol/l of the experimental anti-<italic>TNPO3</italic> duplex
DsiRNAs. TNPO3 mRNA level were detected by quantitative real-time reverse transcription
(qRT-PCR). The data were normalized with GAPDH mRNA and represent the average of three
replicate assays.</p></caption><graphic xlink:href="mtna20126f4"></graphic></fig><fig id="fig5"><label>Figure 5</label><caption><p><bold>Illumina Deep sequence analyses of asymmetric 27-mer <italic>Homo sapiens</italic>transportin 3 (<italic>TNPO3</italic>) Dicer-substrate siRNAs (group II).</bold> HEK 293 cells
were transfected with 10 nmol/l of the asymmetric group II RNA duplexes. Forty
hours post-transfection the total RNAs were isolated and prepared for Illumina Deep
sequencing. The data collection and alignment are described in Materials and Methods
section. (<bold>a</bold>) The total reads and abundance of sense and antisense strands from
each duplex. (<bold>b</bold>) The strand distribution was calculated as the ratio of the
abundance of antisense to sense. The ratio of antisense (AS) to sense (S) is ranked by
the 3′ overhang GG > GC, AA > CC, UU ≫ tt. (<bold>c</bold>) The dicing
pattern L-R and R-L are as previously described. The L-R pattern generates the desired
siRNA species for target knockdown. Total reads from the top 10 antisense strands and
the abundance of L-R cleavage products from the antisense strand. (<bold>d</bold>) Two types
of RNA editing: trimming of the 3′ end and post-transcriptional addition of
nucleotides at the 3′ or 5′ ends.</p></caption><graphic xlink:href="mtna20126f5"></graphic></fig><fig id="fig6"><label>Figure 6</label><caption><p><bold>IC<sub>50</sub> values and strand selectivity of asymmetric 27-mer <italic>Homo
sapiens</italic> transportin 3 (<italic>TNPO3</italic>) Dicer-substrate siRNAs (group II).</bold>(<bold>a</bold>) IC<sub>50</sub> values of the asymmetric 27-mer duplexes were determined
using the psiCHECK assays as described Materials and Methods section. When the antisense
or sense was used as “guide” strand, the target knockdown efficiency is
listed as the IC<sub>50</sub>. (<bold>b</bold>) The strand selectivity was calculated as the
ratio of IC<sub>50</sub> values of the sense to antisense targets.</p></caption><graphic xlink:href="mtna20126f6"></graphic></fig><fig id="fig7"><label>Figure 7</label><caption><p><bold>Illumina Deep sequence analyses of asymmetric 27-mer heterogeneous nuclear
ribonucleoprotein H (<italic>hnRNP H1</italic>) Dicer-substrate small interfering RNA
(siRNAs).</bold> The data collection and alignment were as described above. (<bold>a</bold>)
Total reads of the top 10 sense and antisense strands from each duplex. (<bold>b</bold>) The
strand distribution was calculated as the ratio of the abundance of antisense to sense.
Ratio of antisense (AS) to sense (S) is ranked by the 3′ overhang GG > AA in
site 324 Dicer-substrate small interfering RNAs (DsiRNAs) and GG > CC in site 325
DsiRNAs. (<bold>c</bold>) The dicing pattern L-R and R-L are as previously described. The L-R
model generates the desired siRNA species for target knockdown. The total reads of the
top 10 antisense strands and the abundance of L-R cleavage products are presented.</p></caption><graphic xlink:href="mtna20126f7"></graphic></fig><table-wrap id="tbl1"><label>Table 1</label><caption><title>The 21-mer siRNAs and 27-mer Dicer-substrate siRNAs targeting <italic>TNPO3</italic> and the
target sequences are listed</title></caption><graphic xlink:href="mtna20126t1"></graphic></table-wrap><table-wrap id="tbl2"><label>Table 2</label><caption><title>Illumina Deep sequence analyses and IC<sub>50</sub> values of asymmetric 27-mer
<italic>TNPO3</italic> Dicer-substrate siRNAs (group II)</title></caption><graphic xlink:href="mtna20126t2"></graphic></table-wrap><table-wrap id="tbl3"><label>Table 3</label><caption><title>Deep sequence analyses and IC<sub>50</sub> values of the <italic>hnRNP H1</italic> 25/27-mer
Dicer-substrate siRNAs</title></caption><graphic xlink:href="mtna20126t3"></graphic></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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