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. 2022 Nov 8;3(4):101829.
doi: 10.1016/j.xpro.2022.101829. eCollection 2022 Dec 16.

Million spot binding array platform for exploring and optimizing multiple simultaneous detection events

Michael Gibbons  1 , Jessica M Hong  1 , Mikelle Foster  1 , Mariya Chavarha  1 , Shirley Shao  1 , Llyke Ching  1 , Victoria A Church  1 , Lauren Schiff  1 , Sara Ahadi  1 , Marc Berndl  1 , Phillip Jess  1 , Annalisa Pawlosky  2
Affiliations

Million spot binding array platform for exploring and optimizing multiple simultaneous detection events

Michael Gibbons et al. STAR Protoc. .

Abstract

Large-scale, high-throughput specificity assays to characterize binding properties within a competitive and complex environment of potential binder-target pairs remain challenging and cost prohibitive. Barcode cycle sequencing (BCS) is a molecular binding assay for proteins, peptides, and other small molecules that is built on a next-generation sequencing (NGS) chip. BCS uses a binder library and targets labeled with unique DNA barcodes. Upon binding, binder barcodes are ligated to target barcodes and sequenced to identify encoded binding events. For complete details on the use and execution of this protocol, please refer to Hong et al. (2022).

Keywords: Genomics; High throughput screening; Molecular biology; Sequencing.

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Conflict of interest statement

M.B., P.J., and A.P. are employees and shareholders of Alphabet. Google has filed patent applications related to this work, including PCT/US2020/050574, PCT/US2020/053716, PCT/US2020/040130, PCT/US2020/053715, US20210079398A1, US20210079557A1, US20210102248A1, and WO2021051011A1. All work was completed while authors were affiliated with Alphabet. Current affiliations for authors who have moved on from Alphabet are the following: M.G., 10× Genomics, employee; J.H., University of California, Los Angeles (UCLA); M.F., Conception, employee; M.C., Encodia, Inc., employee; S.S., L.C., University of California, San Francisco (UCSF); V.A.C., Washington University School of Medicine, St. Louis; S.A., Alkahest, employee; L.S., Eikon Therapeutics, employee.

Figures

None
Graphical abstract
Figure 1
Figure 1
Ligating colocalized targets and foundations to pre-existing adapters on the NGS chip
Figure 2
Figure 2
Capturing binding on the NGS chip via the ligation of binder barcodes to foundations In steps 1–4, binder components include the universal bridge (dark green), binder barcode (yellow), and binder tail (light green). In steps 5 and 6, the NGS adapter and bridge are depicted in dark and light blue, respectively.
Figure 3
Figure 3
Composition of BCS target-foundation complex (A) A target-foundation complex assembled on the NGS chip. (B) Magnification of the ligation site between binder tail and foundation. The left-sided DNA strand depicts the 5′ end of the nanobody binder tail (green and yellow) with 5′ phosphorylation (pink circle) and 3′ end of the foundation (orange). The right-sided DNA strand (blue) depicts the universal bridge sequence.
Figure 4
Figure 4
DNA sequences of BCS components (A) The foundation consists of a 16-nt foundation base, 8-nt foundation barcode, and 7-nt bridge-binding sequence. The target tail consists of a 5′ phosphate (represented as “/5Phos/”), 20-nt target base sequence, and a 21- or 25-nt spacer. Red bars symbolize covalent linkage to the target. The forward cololinker consists of 16-nt foundation base complement, 20-nt P7 complement, 44-nt T spacer, and 20-nt hybridization region. The reverse cololinker consists of a 20-nt target base complement, 20-nt P7 complement, 44-nt T spacer, and 20-nt hybridization region. (B) The binder tail consists of a 9-nt ligation spacer, 12-nt binder barcode, 24-nt restriction site spacer (containing an EcoR1 site), and 15-nt T-spacer. Red bars indicate covalent linkage to the nanobody binder. The universal bridge consists of a 5′ region complementary to the restriction site spacer (containing the EcoRI restriction site), a region of 12 nucleotides of 5-nitroindole universal bases (represented as “5”) that can hybridize with any binder barcode, a ligation spacer complement, and a 3′ overhang complementary to the 5′ region of all foundations or previously ligated binder barcode sequences.
Figure 5
Figure 5
NGS chip manual fluid manipulation Fluid is pipetted into the chip through either of the ports, and exits through the other port. The last 5 μL of fluid remains inside the channel.
Figure 6
Figure 6
Example heat map of sequencing counts for binders (horizontal) and their respective targets (vertical), where red indicates high count number and black indicates low count number
See this image and copyright information in PMC

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