Research Citation Collection
Agilent Custom SurePrint Oligonucleotide Libraries

High-Performance Oligonucleotide
Libraries at Your Fingertips!
The Agilent SurePrint Oligonucleotide Library Synthesis platform delivers advanced
and reliable array-based DNA synthesis. We have harnessed our SurePrint platform
to refine the DNA printing process, creating the ability to generate longer oligo
libraries for your important research. Consistent synthesis of high-complexity oligo
pools ensures libraries with superior fidelity and full representation, which improves
functional results and reduces screening time. The fully custom, ultrahigh-quality
oligonucleotide libraries are compatible with any application or experimental
approach.
In this compendium, we have selected ten peer-reviewed publications to illustrate
the utility and versatility of the SurePrint Oligonucleotide Libraries in a variety of
applications. Learn how researchers were able to easily incorporate oligo libraries into
their workflows and successfully move their research forward.

2

Table of Contents
Infectious disease research

De Novo Design of Picomolar SARS-CoV-2 Miniprotein Inhibitors

4

Unbiased Screens Show CD8+ T Cells of COVID-19 Patients
Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside
Outside the Spike Protein

5

Genome-Scale Identification of SARS-CoV-2 and Pan-Coronavirus
Host Factor Networks

6

Functional Interrogation of a SARS-CoV-2 Host Protein Interactome
Identifies Unique and Shared Coronavirus Host Factors

7

Dissection of c-AMP Response Element Architecture by Using Genomic
and Episomal Massively Parallel Reporter Assays

8

Candidate Silencer Elements for the Human and Mouse Genomes

9

A Deep Neural Network for Predicting and Engineering
Alternative Polyadenylation

10

Enhancers research

Mutational Bias and the Protein Code Shape the Evolutionof Splicing Enhancers

11

CRISPR research

Functional Genomics of the Rapidly Replicating Bacterium
Vibrio Natriegens by CRISPRi

12

Gene assembly (DropSynth)

DropSynth 2.0: High-Fidelity Multiplexed Gene Synthesis in Emulsions

13

Massively Parallel reporter
assays3

Infectious disease research

De Novo Design of Picomolar
SARS-CoV-2 Miniprotein Inhibitors
Science, 2020.
doi: http://doi.org/10.1126/science.abd9909

Authors

Abstract

Longxing Cao, Inna Goreshnik,
Brian Coventry, James Brett Case,
Lauren Miller, Lisa Kozodoy, Rita E. Chen,
Lauren Carter, Alexandra C. Walls,
Young-Jun Park, Eva-Maria Strauch,
Lance Stewart, Michael S. Diamond,
David Veesler, and David Baker

Targeting the interaction between the severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting
enzyme 2 (ACE2) receptor is a promising therapeutic strategy. We designed
inhibitors using two de novo design approaches. Computer-generated scaffolds
were either built around an ACE2 helix that interacts with the spike receptor
binding domain (RBD) or docked against the RBD to identify new binding modes,
and their amino acid sequences were designed to optimize target binding,folding, and stability. Ten designs bound the RBD, with affinities ranging from 100
picoMolar to 10 nanomolar, and blocked SARS-CoV-2 infection of Vero E6 cellswith median inhibitory concentration (IC50) values between 24 picoMolar and 35
nanomolar. The most potent, with new binding modes, are 56- and 64-residue
proteins (IC50 approximately 0.16 nanograms per milliliter). Cryo-electron
microscopy structures of these minibinders in complex with the SARS-CoV-2
spike ectodomain trimer with all three RBDs bound are nearly identical to thecomputational models. These hyperstable minibinders provide starting points for
SARS-CoV-2 therapeutics.

Longxing Cao, Inna Goreshnik, Brian Coventry, James Brett Case, Lauren Miller, Lisa Kozodoy, Rita E. Chen, Lauren Carter, Alexandra C. Walls, Young-Jun
Park, Eva-Maria Strauch, Lance Stewart, Michael S. Diamond, David Veesler, David Baker. De novo design of picoMolar SARS-CoV-2 miniprotein inhibitors.
Science, 2020, Oct 23;370(6515):426-431. doi: http://doi.org/10.1126/science.abd9909
4

Infectious disease research

Unbiased Screens Show CD8+ T Cells of
COVID-19 Patients Recognize Shared
Epitopes in SARS-CoV-2 that Largely Reside
Outside the Spike Protein
Immunity, 2020
doi: https://doi.org/10.1016/j.immuni.2020.10.006

Authors

Abstract

Andrew P. Ferretti, Tomasz Kula,
Yifan Wang, Dalena M.V. Nguyen,
Adam Weinheimer, Garrett S. Dunlap,
Qikai Xu, Nancy Nabilsi, Candace R. Perullo,Alexander W. Cristofaro, Holly J. Whitton,
Amy Virbasius, Kenneth J. Olivier, Jr.,
Lyndsey R. Buckner, Angela T. Alistar,
Eric D. Whitman, Sarah A. Bertino,
Shrikanta Chattopadhyay,
and Gavin MacBeath

Developing effective strategies to prevent or treat coronavirus disease 2019
(COVID-19) requires understanding the natural immune response to severe
acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used an unbiased,
genome-wide screening technology to determine the precise peptide sequencesin SARS-CoV-2 that are recognized by the memory CD8+ T cells of COVID-19
patients. In total, we identified 3–8 epitopes for each of the six most prevalent
human leukocyte antigen (HLA) types. These epitopes were broadly sharedacross patients and located in regions of the virus that are not subject tomutational variation. Notably, only 3 of the 29 shared epitopes were locatedin the spike protein, whereas most epitopes were located in ORF1ab or the
nucleocapsid protein. We also found that CD8+ T cells generally do not crossreact with epitopes in the four seasonal coronaviruses that cause the common
cold. Overall, these findings can inform development of next-generation vaccinesthat better recapitulate natural CD8+ T cell immunity to SARS-CoV-2.

Andrew P. Ferretti, Tomasz Kula, Yifan Wang, Dalena M.V. Nguyen, Adam Weinheimer, Garrett S. Dunlap, Qikai Xu, Nancy Nabilsi, Candace R. Perullo,Alexander W. Cristofaro, Holly J. Whitton, Amy Virbasius, Kenneth J. Olivier, Jr., Lyndsey R. Buckner, Angela T. Alistar, Eric D. Whitman, Sarah A. Bertino,
Shrikanta Chattopadhyay, Gavin MacBeath. Unbiased Screens Show CD8+ T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that
Largely Reside outside the Spike Protein. Immunity (2020), 53, 1095–1107. doi: https://doi.org/10.1016/j.immuni.2020.10.006
5Infectious disease research

Genome-Scale Identification of SARS-CoV-2
and Pan-Coronavirus Host Factor Networks
Cell, 2021
doi: https://doi.org/10.1016/j.cell.2020.12.006

Authors

Abstract

William M. Schneider, Joseph M. Luna,
H.-Heinrich Hoffmann,
Francisco J. Sánchez-Rivera,
Andrew A. Leal, Alison W. Ashbrook,
Jérémie Le Pen, Inna Ricardo-Lax,
Eleftherios Michailidis, Avery Peace,
Ansgar F. Stenzel, Scott W. Lowe,
Margaret R. MacDonald, Charles M. Rice,and John T. Poirier

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives ofover one million people worldwide. The causative agent, severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae familyof viruses that can cause respiratory infections of varying severity. The cellular
host factors and pathways co-opted during SARS-CoV-2 and related coronavirus
life cycles remain ill defined. To address this gap, we performed genome-scale
CRISPR knockout screens during infection by SARS-CoV-2 and three seasonalcoronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens
uncovered host factors and pathways with pan-coronavirus and virus-specificfunctional roles, including major dependency on glycosaminoglycan biosynthesis,
sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic
protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well asa requirement for several poorly characterized proteins. We identified an absolute
requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing
protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 andthree seasonal coronaviruses. This human coronavirus host factor compendium
represents a rich resource to develop new therapeutic strategies for acute
COVID-19 and potential future coronavirus pandemics.

William M. Schneider, Joseph M. Luna, H.-Heinrich Hoffmann, Francisco J. Sánchez-Rivera, Andrew A. Leal, Alison W. Ashbrook, Jérémie Le Pen, Inna
Ricardo-Lax, Eleftherios Michailidis, Avery Peace, Ansgar F. Stenzel, Scott W. Lowe, Margaret R. MacDonald, Charles M. Rice, John T. Poirier. Genomescale identification of SARS-CoV-2 and pan-coronavirus host factor networks. Cell 2021, 184, 120-132.
doi: https://doi.org/10.1016/j.cell.2020.12.006
6

Infectious disease research

Functional Interrogation of a SARS-CoV-2
Host Protein Interactome Identifies Uniqueand Shared Coronavirus Host Factors
Cell Host & Microbe, 2021
doi: https://doi.org/10.1016/j.chom.2020.12.009

Authors

Abstract

H. Heinrich Hoffmann, Francisco J.
Sánchez-Rivera, William M. Schneider,
Joseph M. Luna, Yadira M. Soto-Feliciano,
Alison W. Ashbrook, Jérémie Le Pen,
Andrew A. Leal, Inna Ricardo-Lax,
Eleftherios Michailidis, Yuan Hao,
Ansgar F. Stenzel, Avery Peace,
Johannes Zuber, C. David Allis,
Scott W. Lowe, Margaret R. MacDonald,John T. Poirier, and Charles M. RiceThe ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
pandemic has devastated the global economy and claimed more than 1.7
million lives, presenting an urgent global health crisis. To identify host factors
required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed afocused high-coverage CRISPR-Cas9 library targeting 332 members of a recently
published SARS-CoV-2 protein interactome. We leveraged the compact nature ofthis library to systematically screen SARS-CoV-2 at two physiologically relevanttemperatures along with three related coronaviruses (human coronavirus 229E
[HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome
at a much higher resolution than genome-scale studies. This approach yielded
several insights, including potential virus-specific differences in Rab GTPaserequirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, aswell as identification of multiple pan-coronavirus factors involved in cholesterol
homeostasis. This coronavirus essentiality catalog could inform ongoing drug
development efforts aimed at intercepting and treating coronavirus disease 2019
(COVID-19) and help prepare for future coronavirus outbreaks.

H. Heinrich Hoffmann, William M. Schneider, Francisco J. Sánchez-Rivera, Joseph M. Luna, Alison W. Ashbrook, Yadira M. Soto-Feliciano, Andrew A. Leal,
Jérémie Le Pen, Inna Ricardo-Lax, Eleftherios Michailidis, Yuan Hao, Ansgar F. Stenzel, Avery Peace, C. David Allis, Scott W. Lowe, Margaret R. MacDonald,John T. Poirier, Charles M. Rice. Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors.
Cell Host & Microbe 2021, 29(2), 267-280. doi: https://doi.org/10.1101/2020.09.11.291716
7

Massively parallel reporter assays

Dissection of c-AMP Response Element
Architecture by Using Genomic and Episomal
Massively Parallel Reporter Assays
Cell Systems, 2020
doi: https://doi.org/10.1016/j.cels.2020.05.011

Authors

Abstract

Jessica E. Davis, Kimberly D. Insigne,
Eric M. Jones, Quinn A. Hastings,
W. Clifford Boldridge, and Sriram Kosuri

In eukaryotes, transcription factors (TFs) orchestrate gene expression bybinding to TF-binding sites (TFBSs) and localizing transcriptional co-regulators
and RNA polymerase II to cis-regulatory elements. However, we lack a basic
understanding of the relationship between TFBS composition and theirquantitative transcriptional responses. Here, we measured expression driven by
17,406 synthetic cis-regulatory elements with varied compositions of a model
TFBS, the c-AMP response element (CRE) by using massively parallel reporter
assays (MPRAs). We find CRE number, affinity, and promoter proximity largely
determines expression. In addition, we observe expression modulation based on
the spacing between CREs and CRE distance to the promoter, where expression
follows a helical periodicity. Finally, we compare library expression between
an episomal MPRA and a genomically integrated MPRA, where a single cisregulatory element is assayed per cell at a defined locus. These assays largely
recapitulate each other, although weaker, non-canonical CREs exhibit greater
activity in a genomic context.

Davis et al., Dissection of c-AMP Response Element Architecture by Using Genomic and Episomal Massively Parallel Reporter Assays. Cell Systems, 202011, 75–85. Doi: https://doi.org/10.1016/j.cels.2020.05.011
8

Massively parallel reporter assays

Candidate Silencer Elements for the Humanand Mouse Genomes
Nature Communications, 2020
doi: https://doi.org/10.1038/s41467-020-14853-5

Authors

Abstract

Naresh Doni Jayavelu, Ajay Jajodia,
Arpit Mishra and R. David Hawkins

The study of gene regulation is dominated by a focus on the control ofgene activation or increase in the level of expression. Just as critical is theprocess of gene repression or silencing. Chromatin signatures have identified
enhancers, however, genome-wide identification of silencers by computationalor experimental approaches are lacking. Here, we first define uncharacterized
cis-regulatory elements likely containing silencers and find that 41.5% of ~7500
tested elements show silencer activity using massively parallel reporter assay(MPRA). We trained a support vector machine classifier based on MPRA data to
predict candidate silencers in over 100 human and mouse cell or tissue types.
The predicted candidate silencers exhibit characteristics expected of silencers.
Leveraging promoter-capture HiC data, we find that over 50% of silencers areinteracting with gene promoters having very low to no expression. Our results
suggest a general strategy for genome-wide identification and characterization ofsilencer elements.

Doni Jayavelu, N., Jajodia, A., Mishra, A. et al. Candidate silencer elements for the human and mouse genomes. Nat Commun, 2020, 11, 1061.
Doi: https://doi.org/10.1038/s41467-020-14853-5
9

Massively parallel reporter assays

A Deep Neural Network for Predicting andEngineering Alternative Polyadenylation
Cell, 2019
doi: https://doi.org/10.1016/j.cell.2019.04.046

Authors

Abstract

Nicholas Bogard, Johannes Linder,
Alexander B. Rosenberg, and Georg Seelig

Alternative polyadenylation (APA) is a major driver of transcriptome diversity
in human cells. Here, we use deep learning to predict APA from DNA sequencealone. We trained our model (APARENT, APA REgression NeT) on isoform
expression data from over 3 million APA reporters. APARENT’s predictions arehighly accurate when tasked with inferring APA in synthetic and human 3′UTRs.
Visualizing features learned across all network layers reveals that APARENT
recognizes sequence motifs known to recruit APA regulators, discovers
previously unknown sequence determinants of 3′ end processing, and integrates
these features into a comprehensive, interpretable, cis-regulatory code. We
apply APARENT to forward engineer functional polyadenylation signals with
precisely defined cleavage position and isoform usage and validate predictions
experimentally. Finally, we use APARENT to quantify the impact of genetic
variants on APA. Our approach detects pathogenic variants in a wide range of
disease contexts, expanding our understanding of the genetic origins of disease.

Nicholas Bogard, Johannes Linder, Alexander B. Rosenberg, Georg Seelig. A Deep Neural Network for Predicting and Engineering Alternative
Polyadenylation. Cell, 2019, Volume 178, Issue 1, Pages 91-106.e23. doi: https://doi.org/10.1016/j.cell.2019.04.046.
10

Enhancer research

Mutational Bias and the Protein Code Shapethe Evolution of Splicing Enhancers
Nature Communications, 2020
doi: https://doi.org/10.1038/s41467-020-16673-z

Authors

Abstract

Stephen Rong, Luke Buerer, Christy L.
Rhine, Jing Wang, Kamil J. Cygan,
and William G. FairbrotherExonic splicing enhancers (ESEs) are enriched in exons relative to introns andbind splicing activators. This study considers a fundamental question of coevolution: How did ESE motifs become enriched in exons prior to the evolutionof ESE recognition? We hypothesize that the high exon to intron motif ratios
necessary for ESE function were created by mutational bias coupled withpurifying selection on the protein code. These two forces retain certain coding
motifs in exons while passively depleting them from introns. Through the use ofsimulations, genomic analyses, and high throughput splicing assays, we confirm
the key predictions of this hypothesis, including an overlap between protein andsplicing information in ESEs. We discuss the implications of mutational bias asan evolutionary driver in other cis-regulatory systems.

Rong, S., Buerer, L., Rhine, C.L. et al. Mutational bias and the protein code shape the evolution of splicing enhancers. Nat Commun, 2020, 11, 2845.
doi: https://doi.org/10.1038/s41467-020-16673-z
11

CRISPR research

Functional Genomics of the Rapidly
Replicating Bacterium Vibrio Natriegens by
CRISPRi
Nature Microbiology. 2019
doi: https://doi.org/10.1038/s41564-019-0423-8

Authors

Abstract

Henry H. Lee, Nili Ostrov, Brandon G. Wong,
Michaela A. Gold, Ahmad S. Khalil,and George M. Church

The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive
microbial system for molecular biology and biotechnology due to its remarkably
short generation time and metabolic prowess. However, efforts to uncover andutilize the mechanisms underlying its rapid growth are hampered by the scarcity
of functional genomic data. Here, we develop a pooled genome-wide clusteredregularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi)
screen to identify a minimal set of genes required for rapid wild-type growth.
Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered
core genes comprising putative essential and growth-supporting genes thatare enriched for respiratory pathways. We found that 96% of core genes werelocated on the larger chromosome 1, with growth-neutral duplicates of coregenes located primarily on chromosome 2. Our screen also refines metabolic
pathway annotations by distinguishing functional biosynthetic enzymes fromthose predicted on the basis of comparative genomics. Taken together, this work
provides a broadly applicable platform for high-throughput functional genomics
to accelerate biological studies and engineering of V. natriegens.

Lee HH, Ostrov N, Wong BG, Gold MA, Khalil AS, Church GM. Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi.
Nat Microbiol. 2019, Jul;4(7):1105-1113. PMID: 30962569. doi: https://doi.org/10.1038/s41564-019-0423-8.
12

CRISPR research

DropSynth 2.0: High-Fidelity Multiplexed Gene
Synthesis in Emulsions
Nucleic Acids Research, 2020
doi: https://doi.org/10.1093/nar/gkaa600

Authors

Abstract

Angus M. Sidore, Calin Plesa,
Joyce A. Samson, Nathan B. Lubock,and Sriram Kosuri

Multiplexed assays allow functional testing of large synthetic libraries of genetic
elements, but are limited by the designability, length, fidelity and scale of the
input DNA. Here, we improve DropSynth, a low-cost, multiplexed method thatbuilds gene libraries by compartmentalizing and assembling microarray-derived
oligonucleotides in vortexed emulsions. By optimizing enzyme choice, adding
enzymatic error correction and increasing scale, we show that DropSynth canbuild thousands of gene-length fragments at >20% fidelity.

Angus M Sidore, Calin Plesa, Joyce A Samson, Nathan B Lubock, Sriram Kosuri. DropSynth 2.0: high-fidelity multiplexed gene synthesis in emulsions,
Nucleic Acids Research, 2020, Volume 48, Issue 16, 18, Page e95. Doi: https://doi.org/10.1093/nar/gkaa600
13Agilent products are NOT authorized for COVID-19 testing,diagnosis, treatment, or mitigation. Agilent has not validated a
product to detect the novel coronavirus.
For Research Use Only. Not for use in diagnostic procedures.
PR7000-2675
This information is subject to change without notice.
© Agilent Technologies, Inc. 2021
Published in the USA, July 1, 2021
5994-2946EN