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2 results on '"Anna Hieu-Thao Le"'

1. ProtSeq: Toward high-throughput, single-molecule protein sequencing via amino acid conversion into DNA barcodes

Author

Jessica M. Hong, Michael Gibbons, Ali Bashir, Diana Wu, Shirley Shao, Zachary Cutts, Mariya Chavarha, Ye Chen, Lauren Schiff, Mikelle Foster, Victoria A. Church, Llyke Ching, Sara Ahadi, Anna Hieu-Thao Le, Alexander Tran, Michelle Dimon, Marc Coram, Brian Williams, Phillip Jess, Marc Berndl, and Annalisa Pawlosky

Subjects
Biochemistry, Biochemistry applications, Sequence analysis, Proteomics, Transcriptomics, Science
Abstract

Summary: We demonstrate early progress toward constructing a high-throughput, single-molecule protein sequencing technology utilizing barcoded DNA aptamers (binders) to recognize terminal amino acids of peptides (targets) tethered on a next-generation sequencing chip. DNA binders deposit unique, amino acid-identifying barcodes on the chip. The end goal is that, over multiple binding cycles, a sequential chain of DNA barcodes will identify the amino acid sequence of a peptide. Toward this, we demonstrate successful target identification with two sets of target-binder pairs: DNA-DNA and Peptide-Protein. For DNA-DNA binding, we show assembly and sequencing of DNA barcodes over six consecutive binding cycles. Intriguingly, our computational simulation predicts that a small set of semi-selective DNA binders offers significant coverage of the human proteome. Toward this end, we introduce a binder discovery pipeline that ultimately could merge with the chip assay into a technology called ProtSeq, for future high-throughput, single-molecule protein sequencing.

Published
2022
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2. Protseq: An Investigation of High-Throughput, Single-Molecule Protein Sequencing via Amino Acid Conversion into DNA Barcodes

Author

Marc Coram, Diana Wu, Ali Bashira, Shirley Jing Shao, Lauren Schiff, Llyke Ching, Anna Hieu-Thao Le, Brian Williams, Victoria A. Church, Annalisa Pawlosky, Michael Gibbons, Marc Berndl, Mikelle Foster, Phillip Jess, Mariya Chavarha, Ye Chen, Sara Ahadi, Jessica Hong, Zachary Cutts, Michelle Dimon, and Alexander H. Tran

Subjects
chemistry.chemical_classification, Protein sequencing, chemistry, law, Aptamer, Human proteome project, A-DNA, Computational biology, Barcode, Peptide sequence, Systematic evolution of ligands by exponential enrichment, Amino acid, law.invention
Abstract

Despite a need for high-throughput protein sequencing, existing methods are not capable of sequencing the entire human proteome. We demonstrate early progress toward constructing the components of a protein sequencing technology that requires sequential capture and recording of binding over many cycles. The Barcode Cycle Sequencing (BCS) assay uses DNA-barcoded binders to construct a chain of barcodes directly onto a DNA sequencing chip that are used to identify a target. We demonstrated successful barcode capture and DNA-barcode chain sequencing for DNA-DNA and nanobody-protein binding pairs. For DNA-DNA binding, we showed assembly and sequencing of barcodes over 6 consecutive binding cycles. We additionally introduce a binder discovery pipeline called Target-Switch SELEX to discover aptamer binders. Ultimately, we envision merging BCS and Target-Switch SELEX into an end-to-end technology called ProtSeq, a high-throughput, single-molecule protein sequencing method in which DNA-barcoded aptamers read a protein’s amino acid sequence over multiple cycles of terminal amino acid degradation.

Published
2021
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