Compositions and methods of using a synthetic DNase I

Abstract

Compositions and method for making and using a synthetic bovine DNase I are disclosed. More particularly, the sbDNase I of the present invention is a versatile enzyme that cleaves DNA nonspecifically to release 5'-phosphorylated nucleotides. The sbDNase I molecules of the present invention find particular use in a wide range of molecular biology applications, including: degradation of contaminating DNA after RNA isolation; RNA clean-up prior to, or in conjunction with, RT-PCR after in vitro transcription; identification of protein binding sequences on DNA (DNase I footprinting); prevention of clumping when handling cultured cells; tissue dissociation and creation of fragmented DNA for in vitro recombination reactions.

1 - 31 . (canceled)  
     
     
         32 . A synthetic DNase I mutant in which the mutation is replacement of at least one of the amino acids of SEQ ID NO.: 2, SEQ ID NO.: 4 or SEQ ID NO.: 20, with an alternative naturally occurring L-amino acid, the replacement being selected from the group consisting of: (1) a substitution of any of isoleucine, valine, and leucine for any other of these amino acids; (2) a substitution of aspartic acid for glutamic acid or vice versa; (3) a substitution of glutamine for asparagine or vice versa; (4) a substitution of serine for threonine or vice versa; (5) a substitution of glycine for alanine or vice versa; (6) a substitution of alanine for valine or vice versa; (7) a substitution of methionine for any of leucine, isoleucine, or valine and vice versa; and (8) a substitution of lysine for arginine or vice versa, the mutant having DNase I activity in a solution comprising an ionic strength greater that 25 mM.  
     
     
         33 . The mutant of  claim 32 , wherein the replacement is selected from the group consisting of: (1) a substitution of any of isoleucine, valine, or leucine for any other of these amino acids; (2) a substitution of aspartic acid for glutamic acid or vice versa; (3) a substitution of glutamine for asparagine or vice versa; and (4) a substitution of serine for threonine or vice versa and wherein the DNase I comprises a bovine DNase I.  
     
     
         34 . A mutant synthetic bovine DNase I comprising an E13R, an N74K, or an E13R; N74K substitution and one or more replacements for alternative naturally occurring L-amino acids, the one or more replacements being selected from the group consisting of: (1) a substitution of any of isoleucine, valine, and leucine for any other of these amino acids; (2) a substitution of aspartic acid for glutamic acid or vice versa; (3) a substitution of glutamine for asparagine or vice versa; (4) a substitution of serine for threonine or vice versa; (5) a substitution of glycine for alanine or vice versa; (6) a substitution of alanine for valine or vice versa; (7) a substitution for methionine for any of leucine, isoleucine or valine, and vice versa; and (8) a substitution of lysine for arginine or vice versa, the mutant having DNase I activity in a solution comprising an ionic strength of greater than 25 mM.  
     
     
         35 - 95 . (canceled)  
     
     
         96 . An isolated and purified DNase I polypeptide having DNase activity that increases as the salt concentration increases above 10 mM and wherein the DNase I comprises one or more mutations selected from E13R, an N74K, an E13R and an N74K and sequences with 95% sequence homology thereto.  
     
     
         97 . The DNase I of  claim 96 , wherein the DNase I comprises a K m  of less than about 600 nM in a DNase I buffer.  
     
     
         98 . The DNase I of  claim 96 , wherein the DNase I has a Km of about 100 nM in a solution comprising an ionic strength of greater than 25 mM.  
     
     
         99 . The DNase I of  claim 96 , wherein the DNase I has a Km of about 100 nM in an RT-PCR reaction solution.  
     
     
         100 . The DNase I of  claim 96 , wherein the DNase I degrades DNA in a buffer comprising an ionic strength of between about 25 mM to 300 mM.  
     
     
         101 . The DNase I of  claim 96 , wherein the DNase I degrades contaminating DNA for RNA isolation in an RT buffer.  
     
     
         102 . The DNase I of  claim 96 , wherein the DNase I prevents clumping of cultured cells in vitro.  
     
     
         103 . The DNase I of  claim 96 , wherein the DNase I dissociates tissue.  
     
     
         104 . The DNase I of  claim 96 , wherein the DNase I hydrolyzes DNA to create a fragmented library of DNA sequences for in vitro recombination reactions.  
     
     
         105 . The DNase I of  claim 96 , wherein the DNase I removes DNA from non-buffered solutions.  
     
     
         106 . The DNase I of  claim 96 , wherein the DNase I removes DNA and simultaneously with a reverse transcription reaction.  
     
     
         107 . The DNase I of  claim 96 , wherein the DNase I removes DNA from a proteinaceous sample prior to 2-D gel electrophoresis.  
     
     
         108 . The DNase I of  claim 96 , wherein the DNase I has activity in a buffer comprises an ionic strength greater than 50 mM or more and the synthetic DNase I has two-fold more activity that wild-type.  
     
     
         109 . The DNase I of  claim 96 , wherein the DNase I has activity in a buffer with a medium to high ionic strength.  
     
     
         110 . The DNase I of  claim 96 , wherein the DNase I has activity in a buffer with a low concentration of calcium.  
     
     
         111 . The DNase I of  claim 96 , wherein the DNase I has activity in a standard RT-PCR buffer.  
     
     
         112 . The DNase I of  claim 96 , wherein the activity of the DNase I is decreased in high salt concentrations by chelating calcium in the solution.  
     
     
         113 . The DNase I of  claim 96 , wherein the DNase I has activity in a solution selected from the group consisting of an RT buffer, a PCR buffer, a restriction enzyme buffer, or mixtures thereof and the synthetic DNase I maintains two-fold more activity that wild-type DNase I.  
     
     
         114 . The DNase I of  claim 96 , wherein the DNase I comprises a bovine DNase I.  
     
     
         115 . The DNase I of  claim 96 , wherein the amino acid sequence of the DNase I is selected from the group selected from SEQ ID NO.: 2, 4 or 6 and sequences having at least 90% homology therewith.  
     
     
         116 . The DNase I of  claim 96 , wherein the DNase I has activity in a solution comprising about 125 mM NaCl and about 0.5 mM CaCl 2 .  
     
     
         117 . The DNase I of  claim 96 , wherein the DNase I has greater than two-fold higher activity than wild-type in RT buffer; PCR buffer; 50 mM Tris-HCl, 10 mM MgCl 2 , 100 mM NaCl, 1 mM DTT (pH 7.9 at 25° C.); 20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT (pH 7.9 at 25° C.); PBS (+5 mM MgCl 2 ); or 20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate and 1 mM DTT (pH 7.9 at 25° C.) and a reverse transcriptase.  
     
     
         118 . An isolated and purified DNase I polypeptide having a DNase I activity greater than 50% wild-type in a buffer having an ionic strength greater than 25 mM and wherein the DNase I comprises one or more mutations selected from E13R, an N74K, an E13R and an N74K and sequences with 95% sequence homology thereto.