Devices and methods for display of encoded peptides, polypeptides, and proteins on DNA
Inventors
GREENLEAF, WILLIAM J. • Layton, Curtis J.
Assignees
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Publication Number
US-12416100-B2
Publication Date
2025-09-16
Expiration Date
Abstract
A novel method for displaying proteins and peptides is disclosed in which individual proteins or peptides remain associated with the DNA encoding them. Proteins or peptides can be generated by in vitro translation of DNA templates, either free in solution or arrayed on a solid support, such that the proteins or peptides remain immobilized on their DNA templates. In particular, high throughput sequencing can be combined with high throughput functional characterization of encoded proteins and peptides, wherein the identity of each protein or peptide is determined by DNA sequencing, and functional studies are carried out directly on each protein or peptide while immobilized on the DNA template encoding it. The methods of the invention should find numerous applications, for example, in high throughput genetic or pharmacological screening, epitope mapping, and protein engineering and directed evolution.
Core Innovation
The invention relates to DNA-templated protein or peptide display in which ribosomal translation products remain associated with their encoding DNA. A DNA molecule is attached to a surface and comprises a ribosome stall sequence to keep the nascent polypeptide stably associated with the encoding DNA, and the stall sequence includes a polyproline-encoding sequence adjacent to a stop codon.
The ribosome stall sequence further includes an arginine-histidine-arginine tricoding sequence adjacent to the polyproline-coding sequence. The polyproline sequence is positioned on a 5′ side of the stop codon, and the arginine-histidine-arginine tricoding sequence is adjacent to the polyproline-coding sequence, thereby defining an ordered ribosome-stall arrangement within the DNA molecule.
The disclosed approach supports massive parallel arrays on DNA chips and/or flow cells compatible with high-throughput sequencing. Identity of displayed polypeptides or peptides is read by DNA sequencing, while functional assays are performed directly on-displayed proteins or peptides.
The partial content also documents example implementations using a translation-only system to support DNA-associated display, and evidences DNA and protein co-localization on sequencing chips. Functional validation includes on-chip FLAG antibody binding and SNAP-tag activity on sequencing chips, together with quantitative titer or activity patterns.
Claims Coverage
The partial independent claim coverage centers on one independent composition claim with multiple dependent refinements that constrain the stall sequence architecture and the physical context of the DNA attachment. The main inventive features are defined by the specific arrangement of a polyproline-encoding sequence relative to a stop codon and an arginine-histidine-arginine tricoding sequence, together with optional dependent limitations that specify sequence identity, stop-codon options, and surface context.
Surface-attached DNA ribosome stall sequence architecture
A composition comprising a DNA molecule attached to a surface, wherein said DNA molecule comprises a ribosome stall sequence, said ribosome stall sequence comprises a polyproline-encoding sequence adjacent to a stop codon, wherein said polyproline sequence is located on a 5′ side of said stop codon, and an arginine-histidine-arginine tricoding sequence adjacent to said polyproline-coding sequence.
Ribosome stall nucleotide sequence identity to SEQ ID NO: 12
The ribosome stall sequence comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 12.
Polyproline-adjacent stop codon selection
The stop codon is UAG, UAA, or UGA.
5′ side placement of tricoding relative to polyproline-coding sequence
The arginine-histidine-arginine coding sequence is located on a 5′ side of the polyproline-coding sequence.
Microfluidic or flow-cell inner surface attachment context
The surface is an inner surface of a microfluidic device or a flow cell device.
Adapter sequences comprising SEQ ID NO: 1
The adapter sequences comprise the nucleotide sequence of SEQ ID NO. 1.
Overall, the claim set focuses on a DNA molecule attached to a surface that carries a ribosome stall sequence defined by a polyproline-encoding region positioned on the 5′ side of an adjacent stop codon and an arginine-histidine-arginine tricoding sequence adjacent to the polyproline-coding region. Dependent claim refinements further constrain stall nucleotide identity, permitted stop codon types, relative positioning, the inner surface context, and adapter sequence definition using SEQ ID numbers.
Stated Advantages
Supports stable association of ribosomal translation products with their encoding DNA, enabling DNA-associated display of nascent polypeptides.
Enables massive parallel arrays on DNA chips and/or flow cells compatible with high-throughput sequencing for reading identity by DNA sequencing.
Allows functional assays directly on-displayed proteins or peptides, including epitope mapping, immune response profiling, ligand/protein screening, and enzymatic activity assays.
Documented Applications
Massive parallel arrays on DNA chips and/or flow cells for reading identity by DNA sequencing while performing functional assays directly on-displayed proteins or peptides.
Epitope mapping using directly displayed proteins or peptides.
Immune response profiling using directly displayed proteins or peptides.
Ligand/protein screening using directly displayed proteins or peptides.
Enzymatic activity assays using directly displayed proteins or peptides.
Directed evolution workflow using the disclosed display and readout framework.
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