Non-standard amino acid containing compositions and uses thereof
Inventors
Mandell, Daniel J. • GREGG, Christopher J. • STRANGES, Peter B.
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11584921-B2
Publication Date
2023-02-21
Expiration Date
Abstract
Provided herein is a stabilized DNase I polypeptide containing a non-standard amino acid that maintains enzymatic activity even under harsh conditions, such as reducing environments. The stabilized DNase I polypeptide has enzymatic activity in reducing environments that is higher than a corresponding DNase I polypeptide without the non-standard amino acids under the same conditions. Also provided herein are polynucleotides encoding the stabilized DNase I polypeptide, cells for expressing and/or producing the stabilized DNase I polypeptide, and methods of use of the stabilized DNase I polypeptide.
Core Innovation
The invention relates to stabilized deoxyribonuclease I (DNase I) polypeptides comprising at least 90% sequence identity to at least 225 contiguous amino acids of the polypeptide of SEQ ID NO: 1 and from two to four selenocysteines at positions corresponding to 102, 105, 174, and/or 210 of SEQ ID NO: 1. The compositions include one or more diselenide bonds formed by selenocysteines, including a first pair of selenocysteines linked by a first diselenide bond, and in some cases a second pair linked by a second diselenide bond. The DNase I polypeptide is described as stabilized and as retaining higher endonuclease activity and increased stability in reducing environments such as lysis buffers, droplets, and DTT.
The disclosure further describes polynucleotides encoding the stabilized DNase I and expression in bacterial cells, genomically recoded cells, non-bacterial cells, and reassigned codon/tRNA systems for selenocysteine. Expression is linked to reassigned or suppressed codons using selenocysteine tRNA and includes selenolabeled DNase I and GST-seleno-DNase I fusion. The document also describes confirmation of selenocysteine incorporation using mass spectrometry and a higher cleavage rate versus DNase I lacking non-standard amino acids.
Downstream workflows are described as compatible with DNA cleavage for sequencing and single-cell workflows, including vessels, lysis, barcoding, PCR/amplification, and sequencing, with configurable solid supports and primers including barcode primers. The document also describes comparative testing in which the disclosed seleno-DNase digests DNA in high DTT compared with disulfide DNase.
Claims Coverage
The independent claims focus on a DNase I polypeptide composition defined by sequence identity to SEQ ID NO: 1, a defined number and placement of selenocysteines, and the presence of diselenide bonds linking selenocysteines. Three inventive features are consistently present across the claim coverage, with dependent claims tightening the sequence-identity threshold, specifying an additional diselenide-linked pair, and narrowing to recombinant and bacterial-cell embodiments.
DNase I polypeptide with defined selenocysteines and diselenide bonds
A composition comprising a DNase I polypeptide comprising at least 90% sequence identity to at least 225 contiguous amino acids of the polypeptide of SEQ ID NO: 1, wherein the DNase I polypeptide comprises from two to four selenocysteines, wherein at least two of said selenocysteines are located at positions selected from the group consisting of positions corresponding to 102, 105, 174, and 210 of the polypeptide of SEQ ID NO: 1; and wherein the DNase I polypeptide comprises a first pair of selenocysteines linked by a first diselenide bond.
Additional diselenide-linked selenocysteine pair
The composition comprises a DNase I polypeptide that further comprises a second pair of selenocysteines linked by a second diselenide bond.
Tightened sequence identity and recombinant or bacterial embodiments
Dependent claims increase the sequence identity requirement to at least 99% sequence identity to at least 225 contiguous amino acids of SEQ ID NO: 1 and include recombinant DNase I polypeptide and a bacterial cell comprising the composition.
Overall, claim coverage is centered on a DNase I polypeptide composition with high sequence identity to SEQ ID NO: 1 over a contiguous region, incorporation of multiple selenocysteines at specified residue positions, and diselenide-bonded selenocysteine pairs, with dependent claims adding a second diselenide pair, tightening sequence identity, and specifying recombinant and bacterial-cell embodiments.
Stated Advantages
Retains higher endonuclease activity in reducing environments compared with non-modified DNase I.
Increased stability in harsh reducing environments such as lysis buffers and droplets.
Improved thermal stability, indicated by an increased melting temperature (Tm).
Longer protein half-life.
Higher cleavage rate versus DNase I lacking non-standard amino acids.
Seleno-DNase maintains activity in DTT where disulfide DNase does not.
Documented Applications
DNA cleavage for downstream processes including sequencing.
DNA cleavage in workflows compatible with single-cell workflows, including lysis, barcoding, PCR/amplification, and sequencing.
Production and expression contexts including encoding polynucleotides, bacterial cells, genomically recoded cells, non-bacterial cells, and reassigned codon/tRNA systems for selenocysteine.
Interested in licensing this patent?