to recognize centromeric chromatin across different species.
Centromere identity and function are specified by a two-step mechanism wherein the deposition of CENP-A at the centromere by the CENP-A
chaperone HJURP allows for the recognition of
CENP-A by the kinetochore proteins CENP-C
and CENP-N (19). Structures of HJURP (20–22),
CENP-C fragments (5), and CENP-N (this study)
in complex with different forms of CENP-A show
that, in all three cases, only a small number of
residues are involved in the specific recognition
of CENP-A, whereas additional interactions are
used for further stabilization (Fig. 4A, and fig. S11,
C and D). These residues on CENP-A have both
conserved and necessary functions, despite the
variability of their amino acid sequence across
eukaryotes. Our data suggest that the CENP-A–
interacting residues of CENP-N coevolve with
CENP-A, which could reflect a common strategy
for maintaining CENP-A specificity (17). Previous
studies (7, 23) and our results (Fig. 4B and fig. S2,
A and D) show that CENP-N and fragments of
CENP-C that include one of its two CENP-A nucleosome binding domains [fig. S2E, central domain (7, 23), or CENP-C motif (this work)] can
simultaneously bind to the same face of the CENP-A
nucleosome. Furthermore, the kinetochore protein CENP-L has been shown to mediate interactions between CENP-C and CENP-N (9, 23–25).
Thus, the valency of CENP-C/CENP-N/CENP-L
interactions could facilitate clustering of sparse
and nonadjacent CENP-A nucleosomes (Fig. 4C)
(9, 26), which might help to establish the folding
of centromeric chromatin (27, 28) and/or the integrity of the kinetochore (29, 30).
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This research was supported by the intramural programs of the
NCI and NIDDK of the NIH. We thank T. Fox and U. Baxa at
the Center for Molecular Microscopy for help with cryo-EM data
collection, A. Bartesaghi for advice with cryo-EM analysis,
L. Jenkins for mass spectrometry, S. Li for DNA plasmid
purifications, and V. Falconieri for assistance with figure
preparation. This study utilized the computational resources
of the High-Performance Computing Biowulf cluster at the
NIH ( http://hpc.nih.gov). The cryo-EM density map and
atomic coordinates for the hCENP-N1–286/CENP-A nucleosome
complex have been deposited in the Electron Microscopy
Data Bank and the PDB under accession codes EMD-7293 and
6BUZ, respectively. S.C., J.H., A.E.K., Y.B., and S.S. were involved
in all stages of experiment design and interpretation of results;
J.H. prepared nucleosome complex samples and performed
biochemical analysis; H.F. and J.H. performed nuclear
magnetic resonance experiments; R.G. and J.H. performed
analytical ultracentrifugation experiments; H.S. and
A.E.K. performed in vivo experiments; S.C. carried out
cryo-EM grid preparation, data collection, and image
processing; S.C. built the structural model, with input from
J.H.; S.C., Y.B., A.E.K., and J.H. carried out structural analysis;
and S.C., J.H., A.E.K., Y.B., and S.S. integrated all of the
data and wrote the manuscript, with help from all authors. We
declare no competing financial interests.
Materials and Methods
Figs. S1 to S15
20 October 2017; accepted 12 December 2017
Published online 21 December 2017
Multiplexed gene synthesis in
emulsions for exploring protein
Calin Plesa,1 Angus M. Sidore,2 Nathan B. Lubock,1 Di Zhang,3 Sriram Kosuri1,4†
Improving our ability to construct and functionally characterize DNA sequences would broadly
accelerate progress in biology. Here, we introduce DropSynth, a scalable, low-cost method to
build thousands of defined gene-length constructs in a pooled (multiplexed) manner. DropSynth
uses a library of barcoded beads that pull down the oligonucleotides necessary for a gene’s
assembly, which are then processed and assembled in water-in-oil emulsions. We used
DropSynth to successfully build more than 7000 synthetic genes that encode phylogenetically
diverse homologs of two essential genes in Escherichia coli. We tested the ability of
phosphopantetheine adenylyltransferase homologs to complement a knockout E. coli strain in
multiplex, revealing core functional motifs and reasons underlying homolog incompatibility.
DropSynth coupled with multiplexed functional assays allows us to rationally explore sequence-function relationships at an unprecedented scale.
The scale at which we can build and func- tionally characterize DNA sequences sets the pace at which we explore and engineer biology. The recent development of multi- plexed functional assays allows for the facile
testing of thousands to millions of sequences
for a wide array of biological functions (1, 2).
Currently, such assays are limited by their ability
to build or access DNA sequences to test. Natural
or mutagenized DNA sequences (3, 4) allow for
large libraries but are not easily programmed
and thus limit hypotheses, applications, and
engineered designs. Alternatively, researchers
can use low-cost microarray-based oligo pools
that allow for large libraries of designed ~200-
nucleotide (nt) sequences (5), but their short
lengths limit many other applications. Gene syn-
thesis is capable of creating long-length sequences,
but high costs currently prohibit building large
libraries of designed sequences (6–9).
We developed a gene synthesis method we
term DropSynth: a multiplexed approach capable
of building large pooled libraries of designed gene-length sequences. DropSynth uses microarray-derived oligo libraries to assemble gene libraries
at vastly reduced costs. We and others have
developed robust parallel processes to build genes
from oligo arrays, but because each gene must be
assembled individually, costs are prohibitive
for large gene libraries (6, 10). In these efforts,
the ability to isolate and concentrate DNA from
the background pool complexity was paramount
SCIENCE sciencemag.org 19 JANUARY 2018 • VOL 359 ISSUE 6373 343
1Department of Chemistry and Biochemistry, University of
California, Los Angeles (UCLA), Los Angeles, CA, USA.
2Department of Chemical and Biomolecular Engineering,
UCLA, Los Angeles, CA, USA. 3Genomics and Computational
Biology Graduate Group, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA. 4UCLA–
U.S. Department of Energy Institute for Genomics and
Proteomics, Molecular Biology Institute, Quantitative and
Computational Biology Institute, Eli and Edythe Broad Center
of Regenerative Medicine and Stem Cell Research, Jonsson
Comprehensive Cancer Center, UCLA, Los Angeles, CA, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: email@example.com
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