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\begin{document}

\title{PURE mRNA display and cDNA display provide rapid detection of consensus
binding motif via high-throughput sequencing}



\author[1]{Sabrina Galinanes Reyes}%
\author[2]{Yutetsu Kuruma}%
\author[3]{Mai Fujimi}%
\author[4]{Masako Yamazaki}%
\author[4]{Sumie Eto}%
\author[4]{Satoshi Tamaki}%
\author[5]{Asaki Kobayashi}%
\author[6]{Ryo Mizuuchi}%
\author[7]{Lynn Rothschild}%
\author[7]{Mark Ditzler}%
\author[3]{Kosuke Fujishima}%
\affil[1]{University of Glasgow}%
\affil[2]{Japan Agency for Marine-Earth Science and Technology}%
\affil[3]{Tokyo Institute of Technology}%
\affil[4]{MOLCURE, Inc}%
\affil[5]{INSERM}%
\affil[6]{JST}%
\affil[7]{NASA Ames Research Center}%


\vspace{-1em}



  
  \date{June 08, 2024}


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\selectlanguage{english}
\begin{abstract}
The recombinant \emph{in vitro} translation system known as the PURE
system has been used in a variety of cell-free experiments such as
expression of native and \emph{de novo} proteins as well as various
display methods to select for functional polypeptides. We developed a
refined PURE-based display method for the preparation of stable mRNA and
cDNA-peptide conjugates and validated its utility for \emph{in vitro}
selection. Our conjugate formation efficiency exceeded 40\%, followed by
gel purification to allow minimum carry-over of components from the
translation system to the downstream assay enabling clean and efficient
random peptide sequence screening. As a proof-of-concept, we chose the
commercially available anti-FLAG M2 antibody as a target molecule for
validation. Starting from approximately 1.7 x 10\textsuperscript{12}
random sequences, a round-by-round high-throughput sequencing showed
clear enrichment of the FLAG epitope DYKDDD as well as revealing
consensus FLAG binding motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core
FLAG motifs lacking one of the four key residues (DYKxxD) indicates that
Tyr (Y) and Lys (K) appear as the two key residues essential for
binding. Furthermore, comparison between mRNA display and cDNA display
method resulted in overall similar performance with slightly higher
enrichment for mRNA display. The consistency of two different display
methods achieved by commercially available PURE system will be useful
for future studies to explore sequence and functional space of diverse
polypeptides.%
\end{abstract}%



\sloppy


\textbf{Keywords}

mRNA display, cDNA display, PURE system, peptide sequence screening,
FLAG consensus motif



\textbf{Introduction}

Recombinant \emph{in vitro} transcription-translation (TX-TL)
procedures, also known as cell-free systems, have becoming increasingly
common in recent years for a variety of biochemical and molecular
procedures, mainly due to their versatility, and particularly in
synthetic biology (Hammerling, Kr\selectlanguage{ngerman}üger, \& Jewett, 2019; Keasling, 2012;
Perez, Stark, \& Jewett, 2016; Villarreal \& Tan, 2017). These systems
allow flexibility in the reaction condition of parameters for protein
production, such as the transcription and translation machinery, as well
as minimizing the possible limitations that carry working with living
cells (Carlson, Gan, Hodgman, \& Jewett, 2012; Hodgman \& Jewett, 2012;
Silverman, Karim, \& Jewett, 2019). Two main types of cell-free systems
exist: those that derive from cell extracts and those that use purified
recombinant proteins. The cell lysate-based systems were the first to be
developed, and employ extracts from a variety of living organisms such
as, \emph{Escherichia coli} , yeast, fall armyworm, wheat germ, tobacco,
rabbit reticulocytes and HeLa cell line (Anderson, Straus, \& Dudock,
1983; Buntru, Vogel, Spiegel, \& Schillberg, 2014; Ezure, Suzuki, \&
Ando, 2014; Jackson \& Hunt, 1983; Sun et al., 2013; Wang, Zhao, \&
Zhao, 2014; Yadavalli \& Sam-Yellowe, 2015). The application of using
cell lysates quickly gained popularity as it tackled constraints
encountered when performing synthetic biology research using living
organisms, such as expression of protein that exhibits cellular toxicity
or protein production under growth toxic compound (Bowie et al., 2020;
Kay \& Jewett, 2020; Tinafar, Jaenes, \& Pardee, 2019). These systems
often produce high yield of protein products; however, some issues still
remain due to components that actively degrade the mRNA and proteins
(nucleases, proteases), and the presence of additional unknown factors
included in the cell lysate. While ongoing research continues to
mitigate these issues (Didovyk, Tonooka, Tsimring, \& Hasty, 2017;
Fujiwara \& Doi, 2016), cell-free systems using recombinant protein
elements and purified ribosomes such as PURE system (Kuruma \& Ueda,
2015; Y Shimizu et al., 2001; Yoshihiro Shimizu, Kuruma, Kanamori, \&
Ueda, 2014), offer a contaminant-free alternative with a final
significant protein yield (Kazuta, Matsuura, Ichihashi, \& Yomo, 2014).
Lately, more research has focused on simplification, robustness and
low-cost for reconstruction (Lavickova \& Maerkl, 2019) as well as
altering the energy source (Wang et al., 2019) in the PURE system.

These advantages of recombinant systems are particularly beneficial when
one needs a control over conditions for high-throughput screening and
directed evolution of peptide/proteins (Contreras-Llano \& Tan, 2018;
Dodevski, Markou, \& Sarkar, 2015; Fujii et al., 2014). Different
display methods (phage display (Ledsgaard, Kilstrup, Karatt-Vellatt,
McCafferty, \& Laustsen, 2018), yeast display (Boder \& Wittrup, 1997;
Cherf \& Cochran, 2015), ribosome display (Zahnd, Amstutz, \& Plückthun,
2007), liposome display (Fujii, Matsuura, Sunami, Kazuta, \& Yomo,
2013), DNA display (Bertschinger \& Neri, 2004; Doi \& Yanagawa, 1999;
Yonezawa, Doi, Kawahashi, Higashinakagawa, \& Yanagawa, 2003), cDNA
display (Naimuddin \& Kubo, 2016; Yamaguchi et al., 2009), mRNA display
(Nemoto, Miyamoto-Sato, Husimi, \& Yanagawa, 1997; Roberts \& Szostak,
1997; Seelig, 2011)) use various strategies to couple genotype to
phenotype and as such have become indispensable tools for directed
evolution. Among the display methods, \emph{in vitro} approaches such as
mRNA, cDNA, and ribosome display can screen the highest number of
molecules (up to 10\textsuperscript{13}) to be tested because they are
not limited by the efficiency of transformation or transfection. In the
case of mRNA display, screening of large libraries is achieved by
creating a covalent phenotype-genotype linkage between an mRNA and the
polypeptide it encodes using puromycin (Takahashi, Austin, \& Roberts,
2003). Moreover, \emph{in vitro} reactions can be easily modified to
suit a specific environment for functional screening (Josephson,
Ricardo, \& Szostak, 2014).

The utility of mRNA display is limited by the relative instability of
mRNA-protein conjugates, especially in cell lysate-based translation
systems, due to the presence of proteases and especially ribonucleases
(Hino et al., 2008; Opyrchal, Anderson, Sokoloski, Wilusz, \& Wilusz,
2005; Shin \& Noireaux, 2010). The use of RNase inhibitors and
nuclease-free chemicals can help minimize the degradation of RNA
components (Newton, Cabezas-Perusse, Tong, \& Seelig, 2020; Seelig,
2011). The recent advent of reconstituted contaminant-free PURE
translation system has made \emph{in vitro} display methods more popular
for screening antibodies (Kanamori, Fujino, \& Ueda, 2014; Nagumo,
Fujiwara, Horisawa, Yanagawa, \& Doi, 2016) and functional
peptidomimetics (Bashiruddin \& Suga, 2015). Since the PURE system
operates primarily with reconstituted components, it offers increased
stability of mRNA-protein conjugates. In addition, the cDNA display
method, which converts translated mRNA-peptide conjugates into
mRNA/cDNA-peptide conjugates, is advantageous under conditions where RNA
instability is an issue during the selection step, such as targeting
cell surface antigens under the presence of cellular ribonucleases (Ueno
\& Nemoto, 2012; Yamaguchi et al., 2009).

Here, we have established robust PURE system-based mRNA display and cDNA
display methods, and compare their performance to screen for FLAG
epitope sequences against anti-FLAG M2 antibody (Roosild, Castronovo, \&
Choe, 2006). Next-generation sequencing has been recently used along
with display technology to provide an overview of sequence distribution
(Fujimori et al., 2012). Hence, we performed round-by-round deep
sequencing to validate our method by monitoring stepwise enrichment
patterns of the core FLAG epitope motifs and contribution of key
residues that would otherwise be difficult to trace by traditional
sequencing methods.

\textbf{Materials and Methods}

\textbf{Linear DNA library construction}

Linear DNA libraries were constructed by annealing two single stranded
DNA (ssDNA) oligos and extension by Klenow polymerase. Oligos were
ordered through FASMAC Inc., (Supplementary Table 1). A final
concentration of 3 \selectlanguage{greek}μ\selectlanguage{english}M of Display-F and Display-FLAG-random-R oligos were
used for the synthesis of FLAG-random library, as well as Display-F and
Display-10aa-random-R oligos for the synthesis of 10aa-random library
and Display-F and FLAG-control-R oligo for the synthesis of fixed FLAG
(DYKDDDDK) sequence for western blot analysis. Oligo pairs were mixed
with final 200 \selectlanguage{greek}μ\selectlanguage{english}M dNTPs in Klenow buffer (Takara Bio) and annealed by
heating at 92  for 30 seconds followed by cooling to room temperature.
Then 10U of Klenow Fragment (Large Fragment \emph{E. coli} DNA
Polymerase I;l Takara Bio) was added to the reaction mix and an
extension step was performed at 37  for 1 hour, followed by
inactivation of the enzyme at 50  for 15 min. The Linear DNA libraries
(FLAG-random and 10aa-random) were further column purified using the
Wizard SV Gel and PCR Clean-Up System (Promega) and quantified by
Nanodrop2000c (Thermo Fisher Scientific).

\emph{In vitro} \textbf{transcription and DNA-tag ligation}

\emph{In vitro} transcription was performed using HiScribe T7 Quick
High Yield RNA Synthesis Kit (NEB) with approximately 500 ng (11 pmol,
6.6 x 10\textsuperscript{12} molecules) of linear DNA library at 37 
for 6 h according to the manufacturer's protocol. The transcribed RNA
was cleaned up using the NucleoSpin\selectlanguage{ngerman}® RNA Clean-up kit (Macherey-Nagel).
To anneal the mRNA library with a puromycin-FITC DNA tag, a 100 \selectlanguage{greek}µ\selectlanguage{english}L
reaction was set up consisting of final concentrations of 4 \selectlanguage{greek}μ\selectlanguage{english}M
transcribed mRNA, 6 \selectlanguage{greek}µ\selectlanguage{english}M puromycin-FITC DNA tag (Supplementary Table 1)
and 1 mM ATP in T4 ligation buffer (NEB). The reaction was incubated at
90  for 30 sec, and then cooled to room temperature at 1 per second to
correctly anneal the mRNA library with the puromycin-FITC DNA tag. Next,
3U of T4 PNK Kinase (NEB) and 20U of T4 RNA ligase (NEB) were added to
the reaction mixture and further incubated at 25  for 30 min.

\textbf{Gel electrophoresis, electroelution and ethanol precipitation}

Gel electrophoresis of the linear DNA library samples were performed on
a 2\% agarose Tris-Acetate-EDTA (TAE) gel, run at constant 100V.
Transcribed mRNA libraries and their FITC-puromycin-FITC DNA tag ligated
products (mRNA-tag) were resolved using mini size (8 ml) 8 M urea 6\%
polyacrylamide Tris-borate-EDTA (TBE) gel at a constant 50 mA. SYBR Gold
staining (Thermo Fisher Scientific) was used to detect DNA and mRNA
products. For gel excision, ligation product was run on a large-scale
urea-TBE gel (40 ml). A clean scalpel was used to cut off a gel strip
containing mRNA-tag under a blue light LED transilluminator (FUJIFILM
Wako Pure Chemical). Gel purification was performed using standard
electroelution method using a Model 422 Electro-Eluter (Bio-Rad). First,
the gels were crushed into small pieces and placed inside the
Electro-Eluter tubes (Bio-Rad) with membrane cap (12 kDa cut-off)
attached. Tubes were filled with 1x TBE buffer and run at 10 mA/tube for
30 min. Then, the polarity was reversed for 1 min to release the
mRNA-tag from the membrane. Eluted sample solution was recovered and
subjected to ethanol precipitation. For ethanol precipitation, 10\%
sample volume of 3 M sodium acetate was added to the eluted sample
solution, then 3 volumes of 99.5\% ethanol (FUJIFILM Wako Pure Chemical)
was added. After mixing, the was incubated overnight at -20  to help
increase the yield, and the mixture then centrifuged at 4, 20,000 x g
for 60 min. The supernatant was carefully discarded, and 1 ml of 70\%
ethanol was added to the white pellet. A second centrifugation was
performed at 4 , 20,000 x g for 15 min. The supernatant was again
carefully discarded, and the pellet was air-dried at room temperature
for 30 min. Finally, the pellet was resuspended in RNase free water and
quantified by a Nanodrop 2000c.

\emph{In vitro} \textbf{translation using cell-free system for mRNA
display}

Two \selectlanguage{greek}μ\selectlanguage{english}g of the mRNA-tag sample was used for \emph{in vitro} translation
using PUREfrex 1.0 (Gene Frontier) in the 100 \selectlanguage{greek}µ\selectlanguage{english}l reaction mix. The
optimized condition for efficient mRNA-peptide conjugate formation are
as follows: initial translation at 37  for 30 min, followed by addition
of a salt mix to a final concentration of 32.5 mM
MgCl\textsubscript{2}and 375 mM KCl, and then a second incubation at 37
 for 60 min. After this step, an equal volume of 2x Laemmli sample
buffer (Bio-Rad) was added, mixed and centrifuged at 10,000 x g, 1 min
at room temperature to remove the salt precipitant. The supernatant
containing the translated product was recovered and further resolved on
a 3.5\% stacking - 10\% resolving polyacrylamide SDS-PAGE gel at
constant 50 mA. 8 M urea was added to the resolving gel only.
Afterwards, the gel band corresponding to mRNA-peptide conjugate was
excised, purified using the Electro-Eluter. and precipitated with
ethanol, as described in the previous section.

\textbf{Optimization of mRNA-peptide conjugate formation}

The formation rate of mRNA-peptide conjugate using
PUREfrex\selectlanguage{ngerman}\textsuperscript{®}1.0 was optimized through iterations of three
different reaction conditions: translation time, salt mix concentration,
and incubation time after salt mix addition. All reactions were
performed at 10 \selectlanguage{greek}μ\selectlanguage{english}l scale reaction. For the translation time, we tested
0, 5, 15, 30 and 60 minutes at 37 , prior to the addition of salt mix
(final conc, 32.5 mM MgCl\textsubscript{2} and 375 mM KCl) followed by
60 minutes of incubation. For the optimization of salt mix
concentration, we first conducted 30 minutes translation at 37 , then
either added no salt or 1x, 1/2x, 1/4x, 1/8x concentration of 65 mM
MgCl\textsubscript{2} and 750 mM KCl concentration originally reported
to promote covalent bond between puromycin and polypeptide chain
(Naimuddin et al., 2011; Naimuddin \& Kubo, 2016). For the incubation
time after salt addition, we initially performed 30 minutes translation,
added salt to final 32.5 mM MgCl\textsubscript{2}, 375 mM KCl
concentration, then tested 0, 10, 30, 60 and 90 minutes of incubation
time.

\textbf{Trypsin digestion}

Trypsin digestion was carried out by using 75 ng mRNA-tag sample (with
and without translation under optimal condition), 1 mM
CaCl\textsubscript{2}, 50 mM ammonium bicarbonate, and 0.5 \selectlanguage{greek}µ\selectlanguage{english}g of Trypsin
Gold (Promega). Each reaction was incubated at 37  for 30 min and
resolved in a 3.5\% stacking - 10\% resolving polyacrylamide SDS-PAGE
gel, with 8 M urea added to the resolving gel.

\textbf{Reverse transcription for cDNA synthesis}

Reverse transcription (RT) was performed against purified mRNA-peptide
conjugate to obtain mRNA/cDNA duplexes for cDNA display using
PrimeScript One Step RT-PCR Kit Ver.2 (Takara Bio). The initial peptide
library (0th round) was prepared by mixing FLAG-random and 10aa-random
mRNA-peptide conjugate samples to a 1:10,000 molar ratio. Approximately
200 ng (\textasciitilde{}2 x 10\textsuperscript{12} molecules) of the
mRNA-peptide initial library was reverse transcribed in 50 \selectlanguage{greek}µ\selectlanguage{english}l reaction
volume with final 0.4 \selectlanguage{greek}µ\selectlanguage{english}M of RTPCR-R primer in 1 step buffer (Takara Bio)
supplied by the kit. Tubes were incubated at 80  for 30 seconds, then
cooled down to 4 for primer annealing. After, the PrimeScript 1 step
Enzyme Mix (Takara Bio) was added and incubated at 48  for 20 min. We
also prepared RT(--) tube as a negative control without adding the
enzyme mix. After the incubation, both samples were purified using the
NucleoSpin RNA Clean-up XS kit (Macherey-Nagel) and quantified by
Nanodrop.

\textbf{Western blot analysis}

Western blot analysis was performed to confirm the physical linkage of
peptides and their cognate mRNA sequences in the form of mRNA-peptide or
mRNA/cDNA-peptide conjugates. A control mRNA sequence (Supplementary
Table 1) encoding a FLAG epitope (DYKDDDDK) was ligated with
puromycin-FITC DNA tag and translated under optimized condition (see
previous section). Samples were run on a 10\% polyacrylamide SDS-PAGE
gel at constant 30 mA until the samples stack and reach the separating
gel, and at constant 50 mA for additional 40 minutes. The samples in the
gel were semi-dry transferred to a PDVF membrane (Bio-Rad) at constant
110 mA for 60 minutes. Blocking was carried out using the PVDF Blocking
Reagent for Can Get Signal (Toyobo) and, the membrane was incubated
using Anti DYKDDDDK tag monoclonal antibody peroxidase-conjugated
(FUJIFILM Wako Pure Chemical) and Can Get Signal, solution 2 (Toyobo)
with a 1: 12,000 ratio, respectively. The membrane was then revealed
with chemiluminescence using ECL Select Western Blotting Detection
Reagent (GE Healthcare).

\textbf{Selection using anti-FLAG M2 antibody}

First, 40 \selectlanguage{greek}µ\selectlanguage{english}l of Anti-FLAG M2 Magnetic Beads (Sigma-Aldrich) were washed
two times with TBS-T buffer (50 mM Tris HCl, pH 7.4, with 150 mM, NaCl,
1 mM EDTA, 0.2\% Tween 20), using the DynaMag-2 magnetic stand (Thermo
Fisher Scientific). A total of 100 ng of mRNA-peptide initial library or
mRNA/cDNA-peptide conjugate initial library samples were diluted with
500 \selectlanguage{greek}µ\selectlanguage{english}l of TBS-T buffer. Diluted samples were mixed with washed beads and
incubated at room temperature with gentle mixing in a circular rotator
for 1 hour to allow binding. After the incubation, the supernatant was
removed, and the beads were further washed three times with TBS-T and
additional three times with TBS buffer (without Tween 20). Through 1 to
3 round, beads were resuspended in 50 \selectlanguage{greek}µ\selectlanguage{english}l TBS buffer and solutions were
directly applied to the RT-PCR reaction (see next section for detail) to
generate double strand DNA library for the next selection round and
sequencing. For the 4th round samples, we performed elution in a
stepwise manner using FLAG Peptide (Sigma-Aldrich) with increasing
concentrations from 4 \selectlanguage{greek}μ\selectlanguage{english}g/ml to 20 \selectlanguage{greek}μ\selectlanguage{english}g/ml to 100 \selectlanguage{greek}μ\selectlanguage{english}g/ml. Each of the eluted
fractions, along with the remaining beads, were analyzed with RT-PCR.

\textbf{RT-PCR for amplifying selected sequences}

Total 10 \selectlanguage{greek}µ\selectlanguage{english}l of resuspended magnetic beads with bound mRNA-,
mRNA/cDNA-peptide conjugates were used as a substrate for 50 \selectlanguage{greek}µ\selectlanguage{english}l RT-PCR
reaction volume using PrimeScript One Step RT-PCR Kit Ver.2 with 0.4 \selectlanguage{greek}µ\selectlanguage{english}M
RTPCR-F and RTPCR-R primers (Supplementary Table 1). The reaction mix
was incubated at 80  for 30 seconds, then cooled down to 4  for primer
annealing, followed by the addition of PrimeScript enzyme and incubation
at 48  for 20 min to complete the reverse transcription reaction.
Reaction mix was then split into total 8 tubes to recover samples at
different PCR cycles (0, 5, 10, 15, 20, 25, 30 and 35) to check the
amplification efficiency. The PCR condition was set as follows: initial
denaturation at 94 for 60 second, followed by 30 cycles of 94 for 15
seconds, 62 for 30 seconds and 72 for 25 seconds, then final extension
at 68 for 30 seconds. In order to avoid over amplification of DNA, the
reaction samples were run on a 2\% TAE agarose gel stained by SYBR Gold
and the optimal amplification cycle was determined based on the band
intensity and the absence of non-specific amplified products. Finally,
50 \selectlanguage{greek}µ\selectlanguage{english}l RT-PCR reaction was once again conducted with optimal PCR cycles
(20 cycles for round 1 and 2, 15 cycles for round 3) and the product was
purified using Wizard SV Gel and PCR Clean-Up System for the next round
of selection and sequencing.

\textbf{Next-generation sequencing}

Next-generation sequencing (NGS) was conducted using Illumina MiSeq for
every round of DNA library (0 to 4 rounds). Prior to sequence library
preparation, quantification was carried out using Qubit 2.0 Fluorometer
and Qubit 1X dsDNA HS Assay Kit (Thermo Fisher Scientific). A total 50
ng of the DNA library sample was used as an input for the NEBNext Ultra
II DNA Library Prep with Sample Purification Beads (NEB). We also used
NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) (NEB) to
multiplex our samples. The length of prepped DNA Library was determined
by using Agilent 2100 Bioanalyzer (Agilent Technologies), quantified
using Qubit 2.0 Fluorometer and run on MiSeq illumina platform using the
Miseq Reagent Kit v3 600 cycles.

\textbf{Sequence analysis}

Sequence files were retrieved from the MiSeq illumina platform as a raw
FASTQ file. Sequences corresponding to the coding regions were extracted
using a pattern search program written by Perl script. Only the
sequences with an 8 nucleotide perfect match upstream of the start codon
and downstream of the random region are considered as reliable for
downstream analysis. The reliable sequences (in FASTQ format) were
further processed using FASTAptamer-count (Alam, Chang, \& Burke, 2015)
to rank sort sequences based on their read counts. Consensus sequence
logos were created using WebLogo 3 (Crooks, Hon, Chandonia, \& Brenner,
2004) based on the top 50 reads with specific conditions (fixed letter
and positions).

\textbf{Results}

\textbf{Overview of PURE system-based display method}

We have refined and optimized the mRNA and cDNA display method utilizing
the commercially available PUREfrex1.0 system (\emph{in vitro} TX-TL
system with recombinant elements (Y Shimizu et al., 2001; Yoshihiro
Shimizu, Kanamori, \& Ueda, 2005) ) and screened approximately
10\textsuperscript{12} peptide sequences in a single experiment (Figure
1). One of the important features of our refined method is the stability
of the mRNA-tag product during translation via RNase-free PUREfrex and
two separate gel purification steps that assure the high purity of
mRNA-tag for translation as well as peptide-conjugated products.
Elimination of PURE components (RNA, enzyme, polyamine, cofactors, and
ions) from the mRNA and cDNA-peptide conjugates avoids non-specific
interaction among components, peptide conjugates and target during the
downstream binding assay. Anti-FLAG M2 antibody was chosen as a suitable
target to test the performance of our refined display method given the
nature of its short octapeptide FLAG epitope (DYKDDDDK), known crystal
structure (Roosild et al., 2006), and the evidence that previously three
different display methods (phage display, DNA display and ribosome
display) were able to enrich the core FLAG epitope motif (Miceli,
DeGraaf, \& Fischer, 1994; Osada, Shimizu, Akbar, Kanamori, \& Ueda,
2009; Srila \& Yamabhai, 2013; Yonezawa et al., 2003). The overall
procedure takes total approximately 21 hours (mRNA display) or 22 hours
(cDNA display), requiring standard four working days for one round of
selection (Figure 1). High-throughput sequencing was performed after
collecting multiple rounds of DNA library samples.

\textbf{Random DNA library design}

We designed two different random DNA libraries for the purpose of
monitoring the trajectory of various sequences per round. The
FLAG-random library contains fixed codons encoding first five letters of
FLAG epitope (DYKDDDDK) followed by three consecutive degenerate ``RRN''
codons corresponding to seven amino acids (Asn, Lys, Asp, Glu, Ser, Arg
and Gly). Whereas the 10aa-random library consists of ten degenerate
codons harboring degenerate ``VNN'' and ``NNY'' codons corresponding to
16 and 17 different amino acids, respectively (Supplementary Figure 1).
Expected sequence variation of the FLAG-random and 10aa-random library
are 343 and 1.7 x 10\textsuperscript{12}, respectively. By using the
FLAG-random library as a control, we expected to confirm the enrichment
of the full FLAG epitope sequence in the early rounds. On the other
hand, the 10aa-random library enables us to validate the performance of
our display method by exploring large sequence space for finding a suite
of sequences that are able to bind to anti-FLAG M2 antibody. Both DNA
libraries contain an upstream T7 promoter for \emph{in
vitro}transcription, and a ribosome binding site (RBS) for \emph{in
vitro}translation. The leader sequence at the 3' region is complementary
to the puromycin-FITC DNA tag for efficient ligation. The only
difference between the two libraries are the randomized sequence regions
(Supplementary Figure 1).

\emph{In vitro} \textbf{transcription and ligation}

After the DNA library construction, \emph{in vitro} transcription was
performed, followed by column purification and ligation to the
puromycin-FITC DNA tag. Ligation was carried out via the efficient
Y-ligation method (Nishigaki, Taguchi, Kinoshita, Aita, \& Husimi, 1998)
to connect the 3' end of single strand mRNA, and the phosphorylated 5'
end of the DNA tag (Supplementary Figure 2A). Ligated products were run
on 6 \% polyacrylamide TBE gel with 8 M urea to confirm the band shift
after the ligation. Incorporation of the DNA tag was confirmed by SYBR
Gold staining of the mRNA molecule and the FITC fluorescence detection
of the puromycin-FITC DNA tag (Supplementary Figure 2B). The ligation
resulted in approximately 50\% of the mRNA molecules ligated to the DNA
tag. Ligated mRNA-tag product was further gel purified and electroeluted
to eliminate unligated products. The elimination of free DNA tag is
crucial for the downstream translation reaction since puromycin can
interfere with ribosome. Further mRNA-tag was recovered by ethanol
precipitation in a quantity of several \selectlanguage{greek}µ\selectlanguage{english}g.

\textbf{Optimization of conjugate formation during \emph{in
vitro}translation}

Recovered mRNA-tag was subjected to \emph{in vitro} translation using a
commercial cell-free PUREfrex 1.0 kit to form mRNA-peptide conjugates.
PUREfrex is known for its high purity for eliminating nucleases and
proteases and thus ideal for the application for mRNA display. Indeed,
throughout our experiment we never encountered mRNA-tag degradation
during the translation reaction. The formation rate of the mRNA-peptide
conjugate also depends on the incubation conditions during and after the
translation to promote efficient incorporation of puromycin to the
stalled ribosome at the mRNA-DNA boundary. Previous studies have applied
rare codons at the 3' end of mRNA coding region (Nagumo et al., 2016) or
used long linker in the DNA tag (Naimuddin \& Kubo, 2016) to improve the
conjugate formation. In this study, we have adopted both of these
features by placing rare GGA codon at the 3' end and included polyA (18
nt) in the puromycin-FITC DNA tag. By using the gel purified 10aa-random
mRNA-tag as a substrate, we carried out the optimization of conjugate
formation via iteration of three different parameters: translation time,
salt mix (KCl and MgCl\textsubscript{2}) concentration, and incubation
time after salt addition. Translation time reflects the ribosome
loading, translation of the coding region which ends up stalling at the
3' end due to lack of stop codon. We tested translation at 37  from 0
to 60 minutes and found that 5 minutes of translation is sufficient to
reach over 40\% conjugate formation and the extent of conjugation does
not significantly increase with longer incubation times (Figure 2A).
Next, various salt mix concentrations were tested during post
translation incubation. Previously, a high concentration of KCl has been
shown to improve the accessibility of ribosome-bound peptidyl-tRNA to
puromycin (Van Der Mast \& Bloemers, 1973) and has been adopted in the
recent mRNA and cDNA display methods (Naimuddin \& Kubo, 2016; Seelig,
2011; Yamaguchi et al., 2009). We found that without salt addition,
conjugate formation is less than 10\%, however the addition of 32.5 mM
MgCl\textsubscript{2} and 375 mM KCl greatly increased the formation
rate to above 40\% (Figure 2B). Furthermore, we found the incubation
time after salt mix addition to be important for puromycin to react with
peptidyl-tRNA to form covalent link between mRNA and peptide. Here, 60
minutes of incubation at 37 gave the highest yield with a formation
rate of 48.5\% (Figure 2C). With the above optimized condition,
mRNA-peptide conjugates were synthesized, and gel purified from the
SDS-urea polyacrylamide gel. Throughout all conditions we tested, we did
not observe any mRNA degradation nor mRNA-peptide conjugate degradation
during the translation and incubation steps (Figure 2A and 2C).

\textbf{Confirmation of mRNA- and mRNA/cDNA-peptide conjugates}

We confirmed the presence of synthesized peptide on the mRNA- and
mRNA/cDNA-peptide conjugates with two different methods. First, trypsin
digestion (targeting Lys and Arg) was applied to the 10aa-random
mRNA-tag product with and without translation and revealed on a urea-SDS
PAGE. All peptides contain multiple Lys residues at the fixed C-terminus
region (Supplementary Figure 1) adjacent to the DNA tag, hence the
digested product size should become almost equivalent to the mRNA-tag.
As a result, the upper band in the post-translation product disappeared
after 30 minutes of incubation, indicating that the band was indeed
corresponding to the mRNA-peptide conjugate product (Figure 2D).
Alternatively, by using the mRNA-tag product derived from fixed
FLAG-control sequence (Supplementary Figure 1), the presence of
conjugated peptides before and after the cDNA synthesis was confirmed by
western blot analysis (Figure 2E). FITC fluorescence signal from the
membrane transferred samples and chemiluminescence signal via
anti-FLAG-HRP antibody clearly overlap at the same position, indicating
the formation of both peptide conjugates. Note that under the SDS-PAGE
condition, the double-stranded mRNA/cDNA-peptide conjugate migrated
towards the cathode faster than the single-stranded mRNA-peptide
conjugate due to the doubled charge from the phosphate backbone.

\emph{In vitro} \textbf{selection and RT-PCR}

To evaluate the performance of our mRNA and cDNA display method, we
performed a binding assay against anti-FLAG M2 antibody magnetic beads
under neutral pH condition as described in the Materials and Methods
section. For both methods, FLAG-random and 10aa-random conjugate samples
were mixed to a 1:10,000 molar ratio. After the binding step and
repetitive wash step, beads were recovered and directly subjected to
RT-PCR. To avoid the PCR bias via over amplification, we checked the
amplified product every 5 cycles (0 to 35 cycles) to determine the
appropriate cycle for double-strand DNA library synthesis,
(Supplementary Figure 3). Amplified DNA products from both display
methods became visual at the 15th cycle, and were saturated after 20
cycles. As the selection round progressed, band intensity of the 15th
cycle became stronger, implying the quantity of bead-bound conjugate
products had increased over the course of selection. We also observed a
non-specific band appearing around 200 bp, however this band remained
weak throughout multiple rounds, thus we proceeded with the RT-PCR
reaction. PCR was carried out for 20 cycles for the 1st and 2nd round
libraries and 16 cycles for the 3rd round for both display methods.

\textbf{High-throughput sequencing analysis}

High-throughput sequence data was collected from the Miseq system and
parsed through a Perl script to extract the coding region. Extracted
sequences were further analyzed by the FastAptamer software (Alam et
al., 2015) to count and rank the unique sequences based on their number
of reads. We first focused on the top 50 most abundant sequences from
each round of mRNA display to confirm the enrichment of FLAG epitope
during the selection. Sequences from the FLAG-random library (DYKDDxxx)
resulted in a fixed Asp (D) residue at the 6th position immediately
after the first round of selection, followed by positively charged 7th
position with Arg (R) and Lys (K) and a variable 8th position (Figure
3A). After three rounds of selection, sequences derived from the
10aa-random library converge on a conserved core FLAG motif (DYKxxD) in
two different positions, in one case starting at position 2 and in the
other case starting at position 5 (Figure 3B). This binding motif has
been reported in the previous \emph{in vitro} selection studies to have
strong affinity for anti-FLAG M2 antibody (Miceli et al., 1994; Osada et
al., 2009; Srila \& Yamabhai, 2013; Yonezawa et al., 2003). By focusing
on only those sequences with the core FLAG motif, we further found
features in other variable residues such as weakly conserved Glu (E)
immediately before the FLAG motif, (D/L/N) and (D/F/L/N/Y) located
between the conserved Lys (K) and Asp (D) with over 80\% probability
followed by downstream Pro (P) residue (Figure 3C and 3D).

We next compared the enrichment rate of various FLAG motifs detected
through mRNA display with that of cDNA display. The expected frequency
for the full FLAG epitope sequence (DYKDDDDK) in the initial library
(0th round) is 0.29 reads per million, and indeed we were not able to
detect the epitope sequence from the 0th round in our sample of
1,768,526 sequences. Although as the rounds progressed, we observed
continuous enrichment up to 1479 and 198 reads per million for the 3rd
round mRNA and cDNA display, respectively (Figure 4A). When we focused
on the consensus FLAG motif (DYKDDDxx) with fixed Asp (D) residue at the
6th position, we observed marked enrichment of 261,982 and 31,689 reads
per million, suggesting that 6th position is significantly enhances
binding to the anti-FLAG M2 antibody (Figure 4B). For the final 4th
round, we performed competitive elution with the FLAG epitope
octapeptide (DYKDDDDK) by increasing the concentration in a stepwise
manner. The majority of the selected sequences were replaced by
sequences derived from 10aa-random library harboring consensus FLAG
motif: DYK(D/L/N)(D/F/L/N/Y)D located at two different positions (Figure
4C and 4D).

The highest enrichment of consensus FLAG motifs was achieved from
sequences recovered from the eluant with high concentrations of
competitor FLAG peptide (20 or 100 \selectlanguage{greek}μ\selectlanguage{english}g/ml) reaching total 325,917 and
135,913 RPM for mRNA and cDNA display, respectively (Table 1). Under our
selection condition, mRNA display shows higher enrichment rate of the
consensus FLAG motif (average 28.7 fold per round) over cDNA display
(average 19.1 fold per round). Additionally, with mRNA display, the
abundance of the FLAG motif among the sequences that remain bound to the
beads after all of the elution was consistently greater than with cDNA
display (Figure 4 and Table 1, 100 \selectlanguage{greek}μ\selectlanguage{english}g/ml vs beads). These results
suggest that the mRNA/cDNA \emph{conjugate} is more readily eluted form
the beads.

Finally, the influence of the individual FLAG amino acid residues on
antibody binding was evaluated through the comparison of sequences
lacking one of the four residues of the core FLAG motif (DYKxxD).
Enrichment of sequences lacking Tyr (Y) or Lys (K) residues showed
negative enrichment in the first round indicating the importance of
these residues for primary recognition of the anti-FLAG M2 antibody. The
observed result is consistent with the previous selection (Miceli et
al., 1994; Srila \& Yamabhai, 2013; Yonezawa et al., 2003) and obtained
M2 antibody structural data (Roosild et al., 2006).

\textbf{Discussion}

We have established and refined mRNA and cDNA display methods utilizing
the cell-free translation system (PURE system) for efficient and stable
mRNA and mRNA/cDNA-peptide conjugate synthesis and downstream selection
(Figure 1). One of the highlights of our method is the capability of
preventing carry over components through two separate electroelution
steps to ensure the purity of mRNA and mRNA/cDNA-peptide conjugates.
This could be useful if one intends to target the molecules present in
the PURE system such as nucleotides and translation related RNA and
proteins. Also in comparison to affinity beads purification,
polypeptides do not require peptide affinity tags. Performance of the
two display methods was validated by conducting an epitope search from
approximately 10\textsuperscript{12} peptide sequences against the
well-known anti-FLAG M2 antibody (Roosild et al., 2006). A rapid
enrichment of the canonical FLAG epitope: DYKDDDxx after three rounds of
selection and further, was identified with high-throughput sequencing.
This led to new insights into the variable residues, resulting in the
discovery of the consensus FLAG motif sequences; DYK(DLN)(DFLNY)D. This
motif appeared in both of the sites that were able to accommodate the
motif within the restrictions imposed by the library design. Only three
sites in the original library had degenerate codon sequences that can
code for tyrosine, and of those three sites, one is too close to the
C-terminal end of the random region to be part of the FLAG motif.

Two display methods resulted in overall similar performance, showing
consistency of our method but with slightly higher enrichment for mRNA
display over cDNA display. The mRNA display FLAG motif sequences were
slower to elute from the beads than that of cDNA display, possibly
because the RNA-conjugated peptides smaller size allows it access to
more binding sites on the beads than the larger mRNA/cDNA-peptide
conjugates. In contrast, the cDNA display library appears to have more
non-specific binding on the remaining beads.

High-throughput sequencing has become a powerful approach for
various\emph{in vitro} selection and evolution experiments. We show that
round-by-round sequencing and stepwise competitive elution with the FLAG
peptide revealed consensus FLAG motif over other sequence variants by
the 4th and final round. We also observed additional enrichment of
several amino acids adjacent to the FLAG motif in a library dependent
manner (FLAG-random vs 10aa-random) suggesting high-order epistasis
between residues may exist to account for the optimal binding
conformation. The resulting large sequence database also contains
important information regarding the fitness of individual amino acid
residues. By comparing the enrichment pattern of sequences with a single
residue difference, we were able to highlight the most influential
residues important for anti-FLAG M2 antibody recognition. Such patterns
would be useful to further explore the high-affinity epitope candidates,
along with the recent advancement of array based high-throughput and
quantitative protein assay able to discover high-avidity epitope
variants (Layton, McMahon, \& Greenleaf, 2019). Hence, we expect this
method can be applied to broad range of molecular targets to allow rapid
detection of consensus binding motifs.

\textbf{Acknowledgements}

We thank Shota Nishikawa and Hidenori Watanabe of Tokyo Institute of
Technology, and Stephen Fried and Bryan Lanning from Johns Hopkins
University for their valuable comments on the manuscript.

\textbf{Conflicts of Interests}

The authors declare no conflict of interest.

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https://doi.org/10.1093/nar/gng119

Zahnd, C., Amstutz, P., \& Pluckthun, A. (2007). Ribosome display:
Selecting and evolving proteins in vitro that specifically bind to a
target. \emph{Nature Methods} . https://doi.org/10.1038/nmeth1003



\textbf{Figures}

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/329657/articles/456613-pure-mrna-display-and-cdna-display-provide-rapid-detection-of-consensus-binding-motif-via-high-throughput-sequencing}

\textbf{Figure 1. Schematic overview of mRNA and cDNA display using
PUREfrex system.} The system starts with a designed DNA library
(randomized sequences) harboring T7 promoter and a leader sequence that
is complementary to the DNA tag. DNA library is \emph{in
vitro}transcribed to mRNA library and undergo ligation with the
puromycin-FITC DNA tag. Tagged product is then gel purified and
translated using the PUREfrex 1.0 kit. The resulting mRNA-peptide
conjugate is then gel purified. The purified product is then either used
directly in the binding step (mRNA display) or first reverse transcribed
to become an mRNA/cDNA-peptide conjugate (cDNA display). Both products
are used for binding against anti-FLAG M2 magnetic beads and the
selected conjugates are further reverse transcribed and sequenced using
the Illumina MiSeq system. Preparation time (hours) for each step is
shown in red with exception of Miseq sequencing (5 days) carried out
only after all samples were obtained.

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/329657/articles/456613-pure-mrna-display-and-cdna-display-provide-rapid-detection-of-consensus-binding-motif-via-high-throughput-sequencing}

\textbf{Figure 2. Optimization of translation efficiency and
confirmation of mRNA/cDNA-peptide conjugates.} Gel images of the
mRNA-peptide conjugate formation under various translation conditions.
(A) Difference in length of translation before salt addition. Number
indicates different incubation time (in minutes) carried out at 37
degrees C. (B) Difference in salt concentrations with original
concentration (1x) as 750 mM KCl, 65 mM MgCl\textsubscript{2} used in
the previous studies (Naimuddin et al., 2011; Naimuddin \& Kubo, 2016)
(C) Difference in lengths of incubation time (in minutes) after salt
addition. (D) Trypsin digestion of mRNA-tag and mRNA-peptide conjugate
products. (E) Detection of FITC fluorescence signal from the mRNA-tag,
mRNA-peptide conjugate, and mRNA/cDNA-peptide conjugate products (left)
and detection of FLAG epitope sequence using chemiluminescent Western
blotting (right) from the same membrane.

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/329657/articles/456613-pure-mrna-display-and-cdna-display-provide-rapid-detection-of-consensus-binding-motif-via-high-throughput-sequencing}

\textbf{Figure 3. Consensus logo of top 50 abundant sequences from each
selection round of mRNA display.} A round-by-round consensus logo was
created using Weblogo3 (Crooks et al., 2004) using the top 50 most
abundant sequences based on their RPM. Sequence logos derived from
FLAG-random library (A), 10aa-random library (B), core FLAG motif
bearing sequences within the 10aa-random library (C and D) are color
coded based on their charge (negatively charged: red, positively
charged: blue and others: black). For the 4th round, consensus logo was
only created for the sequences eluted with highest FLAG epitope tag
concentration (100 \selectlanguage{greek}μ\selectlanguage{english}g/ml).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. Round-by-round read counts for the enriched FLAG
motifs.} Sequence reads from initial library (0th) to 3rd round
selection, 4th round with competitive elution with FLAG epitope tag at
4, 20, 100 \selectlanguage{greek}μ\selectlanguage{english}g/ml concentration and remaining beads are counted for (A)
DYKDDDDK, (B) DYKDDDxx, (C) xDYK(D/L/N)(D/F/L/N/Y)Dxxx and (D)
xxxxDYK(D/L/N)(D/F/L/N/Y)D sequences. For each round, read counts for
mRNA display method (orange) and cDNA display (dark blue) are presented
side-by-side.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. Round-by-round enrichment of the motifs lacking a
single key residue from the core FLAG motif.} Sequence reads for partial
FLAG motif lacking each of the four key residues first D (yellow), Y
(blue), K (green) and second D (dark red) are shown for each round
during the (A) mRNA display and (B) cDNA display selection.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure1-final/Figure1-final}

\end{center}
\end{figure}\selectlanguage{english}
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\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2/Figure2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure3/Figure3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure4/Figure4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure5/Figure5}

\end{center}
\end{figure}

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\verb`Table1-final.docx`  available at \url{https://authorea.com/users/329657/articles/456613-pure-mrna-display-and-cdna-display-provide-rapid-detection-of-consensus-binding-motif-via-high-throughput-sequencing}

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