Non-standard amino acid containing compositions and uses thereof

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.

1 .- 83 . (canceled) 
     
     
         84 . A method of making a composition comprising a deoxyribonuclease I (DNase I) polypeptide comprising at least 80% 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 the DNase I polypeptide comprises a first pair of selenocysteines linked by a first diselenide bond, wherein at least two of said two to four selenocysteines are located at positions selected from the group consisting of:
 (a) a position corresponding to position 102 of the polypeptide of SEQ ID NO:1,   (b) a position corresponding to position 105 of the polypeptide of SEQ ID NO: 1,   (c) a position corresponding to position 174 of the polypeptide of SEQ ID NO:1, and   (d) a position corresponding to position 210 of the polypeptide of SEQ ID NO:1;   
       wherein the method comprises expressing the DNase I polypeptide in a bacterial cell. 
     
     
         85 . The method of  claim 84 , wherein the DNase I polypeptide further comprises a second pair of selenocysteines linked by a second diselenide bond. 
     
     
         86 . The method of  claim 84 , wherein the DNase I polypeptide comprises a sequence with at least 90% sequence identity to at least 225 contiguous amino acids of the polypeptide of SEQ ID NO: 1. 
     
     
         87 . The method of  claim 84 , wherein the DNase I polypeptide comprises a sequence with at least 95% sequence identity to at least 225 contiguous amino acids of the polypeptide of SEQ ID NO: 1. 
     
     
         88 . The method of  claim 84 , wherein the DNase I polypeptide has at least 90% sequence identity to the polypeptide of SEQ ID NO:1. 
     
     
         89 . The method of  claim 84 , wherein the DNase I polypeptide comprises four selenocysteines, wherein the four selenocysteines are located at the following positions:
 (a) a position corresponding to position 102 of the polypeptide of SEQ ID NO:1,   (b) a position corresponding to position 105 of the polypeptide of SEQ ID NO: 1,   (c) a position corresponding to position 174 of the polypeptide of SEQ ID NO: 1, and   (d) a position corresponding to position 210 of the polypeptide of SEQ ID NO:1.   
     
     
         90 . The method of  claim 84 , wherein the DNase I polypeptide comprises a selenocysteine at a position corresponding to position 102 of the polypeptide of SEQ ID NO:1 linked by a diselenide bond to a selenocysteine at a position corresponding to position 105 of the polypeptide of SEQ ID NO:1. 
     
     
         91 . The method of  claim 84 , wherein the DNase I polypeptide comprises a selenocysteine at a position corresponding to position 174 of the polypeptide of SEQ ID NO:1 linked by a diselenide bond to a selenocysteine at a position corresponding to position 210 of the polypeptide of SEQ ID NO:1. 
     
     
         92 . The method of  claim 84 , wherein the DNase I polypeptide is recombinant. 
     
     
         93 . The method of  claim 84 , wherein the DNase I polypeptide is a modified bovine DNase I. 
     
     
         94 . The method of  claim 84 , wherein the bacterial cell is a genomically recoded bacterial cell. 
     
     
         95 . The method of  claim 84 , wherein the method further comprises purifying the DNase I polypeptide. 
     
     
         96 . The method of  claim 84 , wherein the method comprises culturing the bacterial cell under conditions in which the amino acid sequence of the DNase I polypeptide is expressed. 
     
     
         97 . A method of cleaving or fragmenting a DNA substrate comprising:
 contacting a deoxyribonuclease I (DNase I) polypeptide to a DNA substrate;   wherein the DNase I polypeptide comprises at least 80% 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 the DNase I polypeptide comprises a first pair of selenocysteines linked by a first diselenide bond, and wherein at least two of said two to four selenocysteines are located at positions selected from the group consisting of:
 (a) a position corresponding to position 102 of the polypeptide of SEQ ID NO: 1, 
 (b) a position corresponding to position 105 of the polypeptide of SEQ ID NO: 1, 
 (c) a position corresponding to position 174 of the polypeptide of SEQ ID NO:1, and 
 (d) a position corresponding to position 210 of the polypeptide of SEQ ID NO: 1. 
   
     
     
         98 . The method of  claim 97 , wherein the DNA substrate is genomic DNA. 
     
     
         99 . The method of  claim 97 , wherein the DNA substrate is from a single cell. 
     
     
         100 . The method of  claim 99 , wherein the method comprises forming a plurality of vessels, wherein each vessel of the plurality of vessels comprises:
 (a) a single cell;   (b) the DNase I polypeptide; and   (c) a buffer.   
     
     
         101 . The method of  claim 100 , wherein the buffer is a lysis buffer and the method further comprises lysing the single cell, thereby releasing the DNA substrate from the single cell. 
     
     
         102 . The method of  claim 101 , wherein the method further comprises barcoding the DNA substrate or fragments thereof. 
     
     
         103 . The method of  claim 97 , wherein the DNA substrate is in a buffer or attached to a solid surface.