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\begin{document}

\title{Single-stranded RNA oligonucleotides that recruit endogenous hnRNPA1
enable the targeted reduction of gene expression}



\author[1]{Justin Melendez}%
\author[1]{Ananya Pal}%
\author[1]{Sidharth Puram}%
\author[1]{Robi Mitra}%
\affil[1]{Washington University School of Medicine in Saint Louis}%


\vspace{-1em}



  
  \date{May 27, 2024}


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\selectlanguage{english}
\begin{abstract}
Targeted gene knockdown has become one of the most powerful tools in
molecular biology and holds substantial promise in therapeutic
applications. While existing technologies such as siRNAs, CRISPRi, and
ASOs effectively and specifically reduce gene expression, few can be
used to first discover the genes that influence a particular phenotype
and then directly transition to being used as oligonucleotide
therapeutics. Thus, a tool that could help bridge the gap between target
discovery and the development of therapeutic leads would benefit the
scientific community. Here, we present hnRNPA1 recruiting
oligonucleotides, or AROs, as single-stranded RNA (ssRNA) molecules that
knockdown transcript levels of target genes. AROs target specific
pre-mRNA transcripts via sequence homology and leverage the ubiquitous
and abundant endogenous RNA-binding protein hnRNPA1 to degrade target
transcripts. Using RT-qPCR, we show that AROs effectively knock down
target genes when delivered via a plasmid and expressed using a Pol II
promoter or when delivered directly as single-strand RNAs. Additionally,
as proof of principle, we use a ssRNA ARO to knockdown KRT14 in squamous
cell carcinoma and show reduced invasive potential. We believe AROs fill
an important niche in the scientific toolbox by taking advantage of
endogenous RNA binding machinery for RNA knockdowns.%
\end{abstract}%



\sloppy


\textbf{Single-stranded RNA oligonucleotides that recruit endogenous
hnRNPA1 enable the targeted reduction of gene expression}

\textbf{AUTHORSHIP}

Justin Melendez\textsuperscript{1}, Ananya Pal\textsuperscript{2},
Sidharth V. Puram\textsuperscript{2} Robi D Mitra\textsuperscript{1*}.

\textsuperscript{1}Department of Genetics and Center for Genome Sciences
and Systems Biology, Washington University in St. Louis, St. Louis MO,
63110

\textsuperscript{2}Department of Otolaryngology - Head and Neck Surgery
and Department of Genetics, Washington University in St. Louis, St.
Louis MO, 63110

* Correspondence to: rmitra@wustl.edu

\textbf{GRANTS}

This work was supported by NIH Grants RF1MH117070, RF1MH117070-S1,
U01MH109133 (National Institute of Mental Health) (R.D.M., J.M),
R01GM123203 (National Institute of General Medical Sciences) (R.D.M.).

\textbf{ABSTRACT}

Targeted gene knockdown has become one of the most powerful tools in
molecular biology and holds substantial promise in therapeutic
applications. While existing technologies such as siRNAs, CRISPRi, and
ASOs effectively and specifically reduce gene expression, few can be
used to first discover the genes that influence a particular phenotype
and then directly transition to being used as oligonucleotide
therapeutics. Thus, a tool that could help bridge the gap between target
discovery and the development of therapeutic leads would benefit the
scientific community. Here, we present hnRNPA1 recruiting
oligonucleotides, or AROs, as single-stranded RNA (ssRNA) molecules that
knockdown transcript levels of target genes. AROs target specific
pre-mRNA transcripts via sequence homology and leverage the ubiquitous
and abundant endogenous RNA-binding protein hnRNPA1 to degrade target
transcripts. Using RT-qPCR, we show that AROs effectively knock down
target genes when delivered via a plasmid and expressed using a Pol II
promoter or when delivered directly as single-strand RNAs. Additionally,
as proof of principle, we use a ssRNA ARO to knockdown KRT14 in squamous
cell carcinoma and show reduced invasive potential. We believe AROs fill
an important niche in the scientific toolbox by taking advantage of
endogenous RNA binding machinery for RNA knockdowns.

\textbf{KEYWORDS}

Gene knockdown, RNA therapy, Synthetic biology, hnRNPA1, Splicing
regulatory element, Epithelial-mesenchymal transition, KRT14.

\textbf{INTRODUCTION}

The ability to manipulate RNA expression and splicing is one of the most
powerful tools in research and holds great promise for therapeutic
applications. In basic science, the ability to target and knockdown a
specific gene of interest using tools such as siRNA and CRISPRi has
allowed us to unravel the intricacies of biological pathways, leading to
many new discoveries (Cong et al., 2013; Gilbert et al., 2014; Kampmann,
2018; Mullenders et al., 2009). In medicine, targeted manipulation of
expression and splicing through antisense oligonucleotides (ASOs) has
led to several promising therapeutics. For instance, the FDA has
approved ASO-based treatments for both Duchenne muscular dystrophy, a
child-onset disease in which patients progressively lose muscle function
resulting in wheelchair dependency, the need for ventilation assistance
and ultimately premature death (Aartsma-Rus \& Krieg, 2017), and spinal
muscular atrophy in infants, a disease caused by mutations in SNM1
leading to musculoskeletal wasting and respiratory failure (Stein \&
Castanotto, 2017).

While a variety of technologies exist to knockdown or modify pre-mRNA in
the cell, few options are available that bridge the gap between research
and clinical settings. Tools such as siRNA, lentivirus, and plasmid
expression are frequently employed as screening tools to find
therapeutic targets (Koike-Yusa et al., 2014). However, the end products
of these methods often run into roadblocks as therapeutics themselves.
siRNA therapies are challenged by double-stranded RNA degradation by the
immune system, off-target effects due to improper strand loading, and
toxicity due to oversaturation of the RNAi machinery (Nogrady, 2019).
CRISPR-based technologies require the introduction of the exogenous Cas9
protein, which poses a significant barrier for many applications, and
their off-target effects are still being evaluated for safety (Tycko et
al., 2019). These limitations result in the identification and
characterization of gene targets with technologies that are either
clinically unviable or suboptimal, thus requiring their re-evaluation
with small molecule screens or other therapeutic methods, resulting in
significant delays in translating bench observations to the clinical
setting.

While ASOs have found purchase as therapeutics, they are heavily reliant
on a variety of nucleotide chemistries and backbone modifications to
achieve the desired results (Roberts et al., 2020). ASOs that degrade
their targets are often composed of RNA with central DNA ``gap''
sequences that hybridize with homologous regions of RNAs and trigger
RNase H mediated degradation, whereas sterically hindering ASOs, such as
morpholinos, incorporate morpholine moieties instead of ribose backbones
and modified phosphonodiamidite linkages to evade degradation (Ekker,
2006). Reliance on these chemistries and modifications makes it
difficult to perform large ASO based screens to identify genes that can
be targeted with this modality. For example, this methodology is
incompatible with the production of lentiviral libraries, which infect
cells clonally and thereby can greatly parallelize the search for
candidate molecules through a combination of cell culture and
next-generation sequencing.

A technology that could seamlessly transition between construct-based
screening and the synthesis of biologically functional molecules would
greatly benefit both the research and clinical communities. The ideal
technology would require minimal chemical modifications to transition
from screening construct to lead compound, be compatible with modern
molecular biology techniques, such as plasmid or viral expression, and
would not require exogenous proteins to function. To address this need,
we created hnRNPA1 recruiting oligonucleotides (AROs) to serve as both a
standalone technology and paradigm for using endogenous RNA binding
proteins to manipulate pre-mRNA.

hnRNPA1 is a ubiquitously expressed RNA binding protein with multiple
roles that are still being uncovered; however, the protein is best known
for the role it plays in alternative splicing and RNA processing
(Jean-Philippe et al., 2013). Splice sites are defined by short 5' (GU)
and 3' (AG) sequences flanking introns. While these dinucleotides are
required for splicing, they do not contain enough information to
correctly specify splice junctions. As a result, regulatory proteins
such as SR proteins and heterogeneous nuclear ribonucleoproteins
(hnRNPs) must bind to exonic splice enhancers (ESEs) and exonic splice
silencers (ESSs) respectively to properly define exon-intron boundaries
(Lee \& Rio, 2015) . Binding of hnRNPA1 to an ESS on pre-mRNA blocks the
binding of SR proteins and splicing machinery leading to cooperative
recruitment of additional hnRNPA1 and suppression of the splice site
(Jean-Philippe et al., 2013).

We sought to harness the ability of hnRNPA1 to suppress splice site
selection by recruiting it to targeted pre-mRNA molecules. To do so, we
constructed single-strand RNA molecules herein referred to as hnRNPA1
recruiting oligonucleotides or ``AROs'' (for \emph{A} 1 \emph{r}
ecruiting\emph{o} ligonucleotide). AROs consist of two parts, a short
(20 -- 25 bp) RNA oligonucleotide targeting domain which is
complementary to the target pre-mRNA, and an hnRNPA1 recruiting loop
derived from the HIV ESS 3, which binds to the RNA binding domain RRM1
of hnRNPA1 and recruits the protein (Jain et al., 2017) (Figure 1A). We
hypothesized that, upon recruitment to the target pre-mRNA, hnRNPA1
would displace local SR proteins and splicing machinery (Figure 1B)
resulting in the suppression of regional splice sites, leading to
frameshifts caused by aberrant exon skipping or intron inclusion.

Due to the ubiquitous nature of endogenous hnRNPA1 across cell types,
AROs do not require any exogenous proteins to function. Further, as AROs
do not rely on DNA-RNA hybrids to trigger RNAase H degradation, AROs can
be produced entirely as RNAs within the cell. Finally, their
single-stranded nature and simple mechanism of action allow them to be
transcribed in vivo using a standard Pol II promotor, so they are
compatible with standard lentiviral screening methodologies. In the
following experiments, we demonstrate that AROs can suppress target mRNA
transcripts, are biologically functional, and can be expressed using
standard molecular biology constructs or delivered directly as
single-strand RNA oligonucleotides.

\textbf{RESULTS}

\textbf{AROs knock down KRT14 expression in an hnRNPA1 specific manner.}

To test whether AROs can knock down gene expression in a targeted
manner, we created a 23 base-pair antisense RNA molecule with homology
to exon 7 of the KRT14 gene and fused an hnRNPA1 recruiting loop to
either the 5' end, the 3' end or both sides of the targeting
oligonucleotide. KRT14 was chosen as a knockdown target because it has
been implicated as a driver of the epithelial-mesenchymal transition in
numerous cancers, (Bilandzic et al., 2019, p. 14; Papafotiou et al.,
2016, p. 14) and Exon 7 was chosen due to its small size (47 base-pairs)
and because it contains several motifs for ASF/SF2 exonic splice
enhancer binding, a regulatory element that hnRNPA1 has been shown to
repress (Cartegni et al., 2003; Kashima et al., 2007). As controls, we
also tested an oligonucleotide containing only the 23 bp targeting
domain (i.e. an ``antisense only'' control with no A1 recruiting loop)
and an ARO with a scrambled targeting domain (Fig 2A), which
respectively served to determine whether the targeting oligonucleotide
or hnRNPA1-recruiting loop alone influenced KRT14 expression. Human
keratinocytes, HaCat cells, were chosen as a model cell line for initial
experiments due to their robust expression of KRT14.

Because hnRNPA1 binds cooperatively (Jean-Philippe et al., 2013), we
hypothesized that we would see the greatest knockdown of gene expression
with the ARO containing two flanking hnRNPA1 recruiting loops
(ARL-Oligo-ARL). Consistent with our expectations, we observed a 56\%
knockdown of KRT14 gene expression in ARL-Oligo-ARL compared to the
carrier control (P \textless{}= 0.001), and a 53\% knockdown of
expression compared with the targeting oligo only (Oligo) control (P
\textless{}= 0.01) indicating that the hnRNPA1 recruiting loop was
required for knockdown to occur (Figure 2B). We also saw significant
knockdown of the ARL-Oligo (P \textless{}= 0.05) and Oligo-ARL (P
\textless{}= 0.05) experimental conditions (18\% and 29\% respectively),
when compared to the targeting oligo only (Oligo) control.
Interestingly, we observed a small (15\%) but statistically significant
difference between the targeting oligo only control (Oligo) and the
scrambled targeting oligo control attached to an hnRNPA1 recruiting loop
(scrOligo-ARL) indicating that the presence of the ARL alone may have a
minor knockdown effect on KRT14. Alternatively, it is possible that the
scrambled targeting oligo has off-target effects influencing the
expression of other transcripts which affect the expression of KRT14.

To test whether the effects of the AROs were hnRNPA1 dependent, each
condition was repeated in the presence of two validated siRNAs designed
to knockdown hnRNPA1 (Figure 2B). Concurrent loss of hnRNPA1 with
expression of ARL-Oligo, Oligo-ARL, and ARL-Oligo-ARL caused significant
and complete loss of previously seen knockdown effects on KRT14
transcript (P \textless{}= 0.05, P \textless{}= 0.01 and P \textless{}=
0.01 respectively). In contrast, differences between the control
constructs, scrOligo-ARL and Oligo, and their hnRNPA1 siRNA knocked down
counterparts were not significant.

Taken together, our results demonstrate that AROs targeting KRT14 knock
down mRNA expression in a targeted and hnRNPA1 dependent manner.

\textbf{AROs targeted to TBK1 knock down RNA expression across multiple
locations on exon 14.}

To evaluate whether AROs could knock down the expression of other genes,
we designed AROs against the TBK1 gene (Figure 3A). We targeted exon 14
because it contained multiple ASF/SF2 sites according to ESE finder
(Cartegni et al., 2003) and because its moderate size (122 bp) allowed
us to evaluate multiple AROs targeting the same exon, as diagrammed in
Figure 3A. To investigate whether AROs act in a location-specific
manner, we designed 5 different AROs targeting exon 14 of TBK1 in 20
base pair segments and tested them in HaCat cells. All 5 AROs
significantly knocked down TBK1 expression (P =\textless{} 0.01) when
compared to the mock transfection carrier control. The largest knockdown
was seen in ARL-TBK1-O3-ARL (51\%) and the smallest in TBK1-O5-ARL
(37\%). These results indicate that all AROs targeting exon 14 of TKB1
were effective at gene knockdown.

As a negative control, we included the ALR-Oligo-ARL construct targeting
the KRT14 gene from the previous experiment. As expected, the
KRT14-targeting ARO did not decrease TBK1 transcript levels. Instead, we
observed a significant (P \textless{}= 0.01) increase in TBK1 gene
expression when KRT14 is knocked down. We hypothesize that the increase
in TBK1 expression is a downstream result of perturbation of KRT14
expression levels.

Our experiments do not seem to indicate any preference for targeting
oligo location along the length of the exon as all of the
oligonucleotides that targeted exon 14 of TBK1 showed similar knockdown
effects.

\textbf{AROs can be delivered directly via lipofection as synthesized
oligonucleotides.}

A major appeal of AROs as a potential therapeutic is their small size
and single-stranded nature. Smaller molecules are easier to deliver and
single-stranded RNA is not targeted by cellular mechanisms that actively
seek out double-stranded RNA for degradation. To determine if AROs could
be delivered directly as single-stranded RNA oligonucleotides, we
ordered synthesized AROs targeting KRT14 and tested them in HaCat cells.
Each molecule was designed to contain phosphorothioate bonds between the
first two and last two bases to inhibit exonuclease degradation. The
targeting domain was the same sequence used in the previous plasmid
based knockdown experiment (Figure 1). We tested a single 5' ARL
(ARL-Oligo) as well as ARLs fused to each end of the targeting oligo
(ARL-Oligo-ARL) and compared them to a previously validated siRNA
targeting KRT14 at varying concentrations (Figure 4A).

We observed significant knockdown effects at all concentrations of
ARL-Oligo and anti-KRT14 siRNA conditions, with diminishing returns at
above 100nM. Interestingly, we did not observe significant knockdown
with ARL-Oligo-ARL oligos, despite this configuration being the most
effective in plasmid experiments, until concentrations of 1 \selectlanguage{greek}µ\selectlanguage{english}M. One
possibility for this may be that the ARL-Oligo-ARL may have had
difficulty making it to the nucleus due to its increased size. Another
possibility is that interactions between complementary regions in the
two ARLs on the oligo caused the targeting domain to be less available
or degraded more quickly than its single ARL counterpart.

\textbf{Knocking down KRT14 via AROs in squamous cell carcinoma reduces
cancer migration capability.}

Having demonstrated that we can knock down genes via delivery of
oligonucleotide AROs, we next sought to test if ARO-mediated gene
knockdown could achieve a therapeutic/biologically functional effect.
KRT14 expression has been shown to be important for metastasis and
invasion in several cancers including bladder and ovarian cancers
(Bilandzic et al., 2019, p. 14; Papafotiou et al., 2016, p. 14). SCC9 is
a squamous cell carcinoma line used as a model for
epithelial-mesenchymal transition studies in head and neck cancer (Zhu
et al., 2012). We hypothesized that knocking down KRT14 in SCC9 using
our ssRNA anti-KRT14 ARO would reduce the ability of these cells to
invade surrounding tissues.

To test this hypothesis, we lipofected SCC9 cells with a KRT14 targeting
ARO (ARL-Oligo) or, as a positive control, an siRNA against KRT-14, at
100 nM and assayed RNA expression by RT-qPCR. After 48 hours a near
complete knockdown of KRT14 transcript levels was observed in both
conditions (P \textless{}= .001) (Figure 4B). We also sought to verify
knockdown of Cytokeratin-14, the protein encoded by KRT14. We performed
a western blot (Figure 5C) and observed a 38\% reduction of protein in
the siRNA treated cells (P\textless{}=0.01) and a 24\% knockdown in
cells treated with ssRNA anti-KRT14 ARO (P\textless{}=0.05). While this
knockdown was less than the observed RNA reduction, this is likely due
to two factors: the long doubling time of SCC9 (\textasciitilde{}72
hours) contributing to slow dilution of the protein, and the protein
turning over at a slower rate than the RNA, which is actively being
degraded in both conditions. Additionally, we observed that differences
in protein knockdown between the ARO and siRNA were not significant.

We next sought to assay the invasive potential of SCC9 cells after the
loss of KRT14. 48 hours after lipofection with synthesized
oligonucleotides or siRNAs, cells were seeded into a Boyden chamber to
test their ability to invade into surrounding tissues. This assay uses a
transwell that is sealed on one side with a porous membrane coated with
Matrigel. The transwell is then suspended inside a larger well, and only
invasive cells are able to migrate through the Matrigel. 48 hours after
transfection, cells were seeded and incubated for 24 hours. Cells that
invaded the Matrigel were then stained, observed via light microscopy
(Figure 5B), and counted. We observed a significant decrease in invasive
potential of cells treated with both the anti-KRT14 siRNA control
(P\textless{}=0.05) as well as our ARO (P\textless{}=0.05) compared to
cells treated only with lipofectamine carrier. Cells treated with the
KRT14 siRNA had 39\% fewer cells migrate into matrigel than the carrier
control, while cells treated with the anti-KRT14 ARO had 43\% fewer
cells relative to the carrier (Figure 5A).

The above results support two important conclusions. First that the
knockdown of KRT14 negatively affects the ability of squamous cell
carcinoma to invade surrounding tissues. Second that AROs delivered as
ssRNAs are biologically functional and able to knock down protein levels
in addition to RNA. We believe these experiments show the usefulness of
AROs as novel and useful biological tools for both RNA and protein
perturbation.

\textbf{DISCUSSION}

In this article, we present hnRNPA1 recruiting oligonucleotides (AROs),
a new tool for mRNA knockdown consisting of a short targeting
oligonucleotide and a hnRNPA1 recruiting loop. Through a series of
RT-qPCR experiments targeting KRT14 and TBK1, we have shown that AROs
expressed from plasmids are able to knock down the expression of two
independent genes in a targeted fashion. We have shown that both the
antisense targeting RNA and the hnRNPA1 recruiting loop must be present
for an efficient knockdown. We have also shown that AROs function in an
hnRNPA1 dependent manner. Finally, we have shown that AROs are
functional in multiple cell lines including human keratinocytes (HaCat)
and squamous cell carcinoma cells (SCC9) and can affect a
loss-of-expression functional phenotype.

Our results suggest that the location of the sequence targeted by an ARO
on the exon of interest does not have a large influence on knockdown
efficiency. We initially hypothesized that the targeting location would
have a bigger impact on ARO function. Specifically, we expected that it
would be necessary to direct AROs to splice junctions or to ASF/SF2
motifs, as preventing binding of SR splice enhancer proteins is a known
mechanism of hnRNPA1 splice site repression (Jean-Philippe et al., 2013;
Kashima et al., 2007). One possible explanation is the cooperative
recruitment of hnRNPA1 molecules along RNA in the 3' to 5' direction
(Jean-Philippe et al., 2013). This might mean that an ARO binding at a
downstream exonic sequence is sufficient to suppress SR protein
recruitment at upstream regions. Another possibility is that the exon we
choose was too small to see location specific effects. Larger exons with
more space between splice sites should be tested in the future to help
answer this question.

A major advantage of AROs over most knockdown technologies is the ease
with which they can transition from promotor-based expression (i.e. from
a plasmid or lentivirus construct) to RNA oligonucleotides that can be
directly delivered to cells or organisms. Here we have shown the utility
of AROs as a potential therapeutic by synthesizing single-stranded RNA
AROs and showing that they are able to achieve similar levels of
knockdown as their plasmid counterparts. By using ssRNA AROs to knock
down KRT14 in SCC9, a squamous cell carcinoma line used as a model for
epithelial-mesenchymal transition studies in head and neck cancer, we
demonstrate that ARO-based knockdown resulted in a biologically relevant
phenotype -- the decreased ability of carcinoma cells to migrate in a
cell invasion assay.

An unexpected finding in our RNA oligonucleotide experiments was that
while the double ARL ssRNA (ARL-Oligo-ARL) worked to some degree at high
concentrations, the single ARL (ARL-Oligo) achieved significantly better
gene knockdown at lower concentrations. This contrasted with our plasmid
experiments which found that double ARL AROs performed best. A possible
explanation for this may be that the additional size or complexity of
double ARL oligonucleotides may have lessened their ability to make it
to the nucleus. Another possibility is that complementary regions
between the two ARLs caused secondary structure leading to
double-stranded portions of the construct being degraded in the
cytoplasm. This would be less likely to occur in the plasmid construct
as those AROs would be produced in the nucleus and would presumably have
more opportunity to affect pre-mRNA before being exported and degraded.

We hypothesize that AROs knock down gene expression by causing errors in
pre-mRNA splicing followed by nonsense-mediated decay. However, it is
important to point out that there are several plausible alternative
hypotheses. For example, hnRNPA1 has been shown to bind to the 3' UTR of
some genes, and this can lead to mRNA deadenylation and degradation
(Geissler et al., 2016). This may be why binding along any point of an
exon in pre-RNA seems to be effective. While more work will need to be
done to determine the specific mechanism of action of AROs to better
understand and improve their function, their utility as biological tools
in their current form remains substantial.

AROs represent a novel paradigm for recruiting RNA binding proteins to
pre-mRNAs via synthetic constructs. Here we have shown that hnRNPA1 can
be recruited via a short targeting oligonucleotide to knock down
transcript levels of a target gene. While more work will need to be done
to understand the exact mechanism of how AROs function, the work here
illustrates their potential as a novel, biologically functional way to
knock down target transcripts through the redirection of endogenous
cellular machinery. Because RNA binding proteins are ubiquitous and
abundant across cell types, their recruitment to targeted pre-mRNAs may
be a useful way to modulate RNA production, modification, or
degradation. The diverse array of functions they perform and their
directable nature make them an ideal source to mine for molecular
biology tools. The ability to express AROs, and similarly designed
molecules, from a Pol II promoter also has substantial advantages. AROs
could be placed under the control of cell or tissue specific promotors
and used in tissue specific studies or placed behind activatable
promotors.

AROs can both be expressed by constructs or used directly as ssRNA
molecules with little modification. This dual functionality puts them in
a unique position to bridge the gap from high-throughput screens to
independently functional molecules. We believe AROs fill an important
niche in the scientific toolbox and we hope the work here can serve as a
template for future constructs that make use of the potential of RNA
binding proteins as tools for the modulation of RNA homeostasis.

\textbf{MATERIALS AND METHODS}

\textbf{Cell culture}

HaCat cells were cultured in Dulbecco's Modified Eagle Medium (Gibco)
with 10\% FBS at 37 degrees Celsius at 5\% CO2 and maintained in flat
bottom T75 flasks. SCC9 cells were cultured in a 3:1 mixture of Ham's
F12 Medium (Gibco) and Dulbecco's Modified Eagle Medium supplemented
with 10\% FBS and maintained at 37 degrees Celsius at 5\% CO2 in T75
flasks. For ARO RT-qPCR experiments, cells were grown in 24 well plates
and lipofected with 500 ng of plasmid using lipofectamine 3000
(Thermofisher). In knockdown experiments, two independent siRNAs (Life
Technologies, Silencer Select Pre-Designed siRNA) were co-transfected at
a final concentration of 10 nM during lipofection, and knockdown of the
target gene was verified via RT-qPCR. In experiments involving direct
delivery of single-stranded RNA, cells were grown in 24 well plates and
lipofected using lipofectamine 3000 (Thermofisher) at the concentrations
listed. For Western blot and Boyden chamber experiments, cells were
maintained in 6 well plates prior to seeding.

\textbf{Boyden Chamber Invasion Assays}

20,000 SCC9 cells were suspended in 200 ul of serum-free growth media
and seeded inside a matrigel-coated transwell. 750 ul of growth media
containing 10\% FBS was added to the larger well. Cells were allowed to
migrate through the pores to the other side of the membrane by
incubating them at 37 degrees Celsius for 24 hours. After 24 hours of
incubation, the transwells were washed in PBS, fixed in 100\% Methanol
for 2 minutes, and then stained in crystal violet solution for 20
minutes. Excess crystal violet was washed off by dipping the transwells
in water. The transwells were then left to dry following which they were
imaged in a brightfield microscope. A custom Image J script was used to
count cells that migrated into the Matrigel coated side of the
transwell.

\textbf{Western Blots}

Cells were harvested 48 hours after lipofection using RIPA buffer and
total cellular protein was extracted. A BCA assay (Pierce BCA Protein
Assay Kit) was used to determine the protein concentration of each
sample. 5 ug of lysate from each sample was loaded into a Mini-PROTEAN
TGX stain-free precast gel (Bio-Rad) and resolved. Once proteins were
resolved, gels were activated on a ChemiDoc (Bio-Rad) imager and were
then transferred to a polyvinylidene difluoride (PVDF) membrane.
Membranes were probed with mouse Cytokeratin 14 antibody primary
antibody (Life Technologies) and HRP conjugated goat anti-mouse
secondary antibody (Santa Cruz Biotechnology). Total proteins and
chemiluminescence was imaged on the ChemiDoc and Image Lab Software
(Bio-Rad) was used to analyze protein abundance.

\textbf{Statistical analysis}

A Student's t-test was used to test for significance between groups.
Experiments were performed in at least two replicates unless otherwise
stated and error bars show plus and minus standard deviation. P values
of \textless{}= 0.05 were determined to be significant.

\textbf{RT-qPCR}

Cells were grown for 48 hours after lipofection before being harvested
using RNAprotect (Qiagen). Total RNA was extracted using Direct-zol RNA
microprep kit and quantified. Each condition was performed in triplicate
using 50 ng of total RNA. For each 20 ul reaction, RNA was added to Luna
Universal One-Step RT-qPCR master mix (New England Bio Labs) and two
RT-qPCR primers at a final concentration of 0.4 \selectlanguage{greek}µ\selectlanguage{english}M. The following
amplification cycle was run on a QuantStudio 3 real-time PCR machine
(ThermoFisher Scientific): 55 \selectlanguage{ngerman}°C for 10 min, 95 °C for 1 min, followed
by 95 °C for 10 sec and 60 °C for 1 min for 40 cycles. All genes were
normalized to beta Actin (ACTB) and expression was determined via
relative quantification to lipofectamine only carrier control. Gene
specific primers for each gene of interest were ordered from Integrated
DNA Technologies and are as follows: ACTB: 5'-CACCATTGGCAATGAGCGGTTC-3'
and 5'-AGGTCTTTGCGGATGTCCACGT-3', TBK1: 5'-GGATCACTGCCATTTAGACCC-3' and
5'-CAGGCATGTCTCCACTCCAG-3' (PrimerBank 309747068c3), KRT14: 5'-
TGAGCCGCATTCTGAACGAG-3' and 5'-GATGACTGCGATCCAGAGGA-3' (PrimerBank
83641893c1), HNRNPA1: 5'-TCAGAGTCTCCTAAAGAGCCC-3' and
5-ACCTTGTGTGGCCTTGCAT-3' (PrimerBank 83641893c1).

\textbf{Plasmids and Cloning}

A sequence containing two BSMBI restriction sites flanked by hnRNPA1
recruiting loops (ARLs) was cloned into PCDNA 3.1+ (Invitrogen) behind
the CMV promotor between NheI and XbaI restriction sites, upstream of
the polyadenylation sequence to create a Golden Gate vector for ARO
constructs. This vector was then used to clone all additional ARO
constructs except for scrOligo-ARL, Oligo, ARL-Oligo, and Oligo-ARL
(Figure 2A) which were cloned into the same locus via Gibson assembly.
All Golden Gate cloned constructs were ordered as two single-strand DNA
oligos (Integrated DNA Technologies) and annealed such that overhangs
were compatible with the Golden Gate ARO vector. Once cloned, these
annealed oligos served as the targeting oligos for the AROs.
Single-strand RNA ARO oligos were ordered directly from IDT (Integrated
DNA Technologies) and contained two phosphorothioate bonds at each end
to protect the RNA from degradation by exonucleases. The sequence used
for the ARL was as follows: 5'- GGATCCATTCGTAGGAGGAACGGATCC-3'.
Sequences for targeting oligos in AROs used in experiments are as
follows. ARL-Oligo-KRT14 -ARL: 5'CCAGAGGAGAACTGGGAGGAGG-3',
scrambledARL-Oligo: 5'-TGTGGCGAGTAGACTCGAAG-3', ARL-TBK1-O1-ARL:
5'-GTTCCCTGAGAACTGGAAAG-3', ARL-TBK1-O2-ARL: 5'-CCTGAAGACTGGTTTCTATT-3',
ARL-TBK1-O3-ARL: 5'- TCTGCCAGTGATCCACCTGG-3', ARL-TBK1-O4-ARL: 5'-
CTTCTTGATGTGCCCATGCG-3', ARL-TBK1-O6-ARL: 5'- TCTGTCTTTCGGATGAGTGC-3'.

\textbf{AUTHOR CONTRIBUTIONS}

Justin Melendez conceived of the project and performed all lipofections,
cell culture, RT-qPCR, Western-blots, and data analysis and wrote the
initial draft of the manuscript. Sid Puram designed and interpreted and
Ananya Pal designed, performed, and interpreted the Boyden chamber
assays and both reviewed the manuscript. Rob Mitra contributed to the
direction of the project and reviewed the manuscript.

\textbf{ACKNOWLEDGMENTS}

This work was supported by NIH Grants RF1MH117070, RF1MH117070-S1,
U01MH109133 (National Institute of Mental Health) (R.D.M., J.M),
R01GM123203 (National Institute of General Medical Sciences) (R.D.M.).
We thank Tamara Abou-Antoun and Dr. Christian Shively for their
assistance with western blots. We thank the Baldridge Lab at Washington
University in Saint Louis for the generous use of their RT-qPCR machine.

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\textbf{FIGURE LEGENDS}

\textbf{Figure 1:} Overview of hnRNPA1 recruiting molecules
(AROs).\textbf{(A)} Each ARO consists of two parts, a 20 bp hnRNPA1
recruiting loop derived from HIV exon splicing silencer 3, which
recruits hnRNPA1 by binding to RNA binding domain RRM1, and an antisense
targeting domain consisting of 20-25 base pairs of RNA complementary to
a sequence on a target pre-mRNA. \textbf{(B)} The hypothesized mechanism
of AROs. 1. In the absence of AROs splicing occurs normally. 2. The
binding of an ARO to its pre-mRNA target recruits hnRNPA1 and displaces
SR proteins and spliceosome machinery (U2, U2AF65/35, and U1). 3. We
hypothesize this causes repression of splicing resulting in exon
skipping or intron inclusion. This would cause frameshifts in the mRNA
leading to the presence of premature termination codons (PTCs) in the
reading frame upstream of exonic junction complexes (EJCs). UPF1 would
then recognize these sites as mis-spliced mRNA and degrade the RNA by
non-sense mediated decay.

\textbf{Figure 2:} Plasmid expressed AROs knock down KRT14 in human
keratinocytes (HaCat cells). \textbf{(A)} Visual representation of each
ARO and controls. 1. scrOligo-ARL is a scrambled targeting oligo with an
intact hnRNPA1 recruiting loop (ARL). 2. Oligo is a targeting oligo with
homology to Exon 7 of KRT14. 3. ARL-Oligo is the targeting oligo with an
ARL on the 5' end of the construct relative to the targeting domain. 4.
Oligo-ARL is the targeting oligo with an ARL on the 3' end of the
construct relative to the targeting domain. 5. ARL-Oligo-ARL is a
construct with ARLs attached to both the 5' and 3' ends of the targeting
domain. \textbf{(B)} Fold change of KRT14 transcript expression relative
to a carrier only lipofectamine control RT-qPCR. Cells were lipofected
with plasmid for 48 hours and then harvested for RNA extraction. Green
bars indicate plasmid experiments while blue bars indicate experiments
with siRNA designed to knock down hnRNPA1. Three replicates were
performed. Error bars show standard deviation between replicates. A
Student's t test was used to determine significance.

\textbf{Figure 3:} Plasmid expressed AROs targeting exon 14 knock down
TBK1 RNA expression in human keratinocytes (HaCat cells). All AROs were
expressed with ARLs attached to the 3' and 5' ends of the targeting
oligo. \textbf{(A)} Location of targeting oligo homology to exon 14 of
TBK1. \textbf{(B)} Fold change of TBK1 transcript expression relative to
a carrier only lipofectamine control RT-qPCR. Cells were lipofected with
plasmid for 48 hours and then harvested for RNA extraction. Three
replicates were performed. Error bars show standard deviation between
replicates. A Student's t-test was used to determine significance.

\textbf{Figure 4:} Synthesized single-strand RNA oligonucleotides knock
down KRT14 expression across two cell types. (A) Synthesized ssRNAs with
phosphorothioate bonds on the first two and last two bases administered
via lipofection to HaCat cells at varying concentrations. ARL-Oligo-ARL
refers to a ssRNA with two hnRNPA1 recruiting loops on either side of
the targeting domain. ARL-Oligo is composed of a single hnRNPA1
recruiting loop fused to the 5' end of an RNA targeting sequence. A
previously validated siRNA against KRT14 was used as a positive control
at each concentration. Cells were harvested 48 hours post lipofection
and fold change was determined relative to a lipofectamine carrier only
control. (B) The single ARL-Oligo ssRNA and siRNA targeting KRT14 were
lipofected into SCC9 cells. As previously, cells were harvested 48 hours
post lipofection and fold change was determined relative to a
lipofectamine carrier only control. Three replicates were performed in
each experiment. Error bars show standard deviation between replicates.
A Student's t-test was used to determine significance.

\textbf{Figure 5:} AROs show a functional phenotype in a cancer invasion
model. \textbf{(A)} As previously, siRNA targeting KRT14 and an ssRNA
ARO with a single hnRNPA1 recruiting loop fused to the 5' end of a KRT14
targeting oligo were administered at 100 nM via lipofection in SCC9
cells and grown for 48 hours. 20,000 cells were seeded inside a
matrigel-coated transwell for a Boyden chamber invasion assay and
allowed to migrate through the pores to the other side of the membrane
for 24 hours. Cells were then stained with crystal violet and invading
cells were imaged and counted. Two replicates were preformed of this
experiment. Error bars show standard deviation between replicates. A
Student's t test was used to determine significance.
\textbf{(B)}Representative images of crystal violet stained invading
cells in each condition. \textbf{(C)} Protein abundance of KRT14 in
cells lipofected with either ARO (ssRNA ARL-Oligo) or anti-KRT14 siRNA
at a final concentration of 100 nM. Each condition was performed in
triplicate and total protein was harvested 48 hours after lipofection.
Western blots were preformed using KRT14 mouse primary antibody and an
anti-mouse HRP conjugated secondary antibody, and total protein load
using a stain free gel was used as a loading control for normalization.\selectlanguage{english}
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