US2018312822A1PendingUtilityA1

Mmlv reverse transcriptase variants

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Assignee: 10X GENOMICS INCPriority: Apr 26, 2017Filed: May 9, 2018Published: Nov 1, 2018
Est. expiryApr 26, 2037(~10.8 yrs left)· nominal 20-yr term from priority
Inventors:Josephine LeeSamuel MarrsGeoffrey McdermottFrancesca MeschiLuz MontesclarosKatherine PfeifferJoseph Francis ShugaJessica Michele TerrySolongo Batjargal Ziraldo
C12N 9/1276C12Q 1/6806C12N 15/1096C12Y 207/07049C12Q 1/6844C12N 15/1065
53
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Claims

Abstract

Disclosed herein, are compositions, methods, and kits comprising engineered reverse transcription enzymes that exhibit several desired properties such as thermal stability, processive reverse transcription, non-templated base addition, and template switching ability. The engineered reverse transcription enzymes described herein demonstrate unexpectedly higher resistance to cell lysate inhibition, greater ability to capture full-length mRNA transcripts, and demonstrate improved results in small reaction volumes as compared to other engineered reverse transcription enzymes.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . An engineered reverse transcription enzyme, comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 3, wherein said amino acid sequence comprises:
 (i) a truncation of at least 15 amino acids from the N-terminus relative to SEQ ID NO: 3; and   (ii) one or more mutations selected from the group consisting of an E69 mutation, an L139 mutation, a D200 mutation, an E302 mutation, a T306 mutation, a W313 mutation, a T330 mutation, an L435 mutation, a P448 mutation, a D449 mutation, an N454 mutation, a D524 mutation, an L603 mutation, and an E607 mutation relative to SEQ ID NO: 3.   
     
     
         2 . The engineered reverse transcription enzyme of  claim 1 , wherein said one or more mutations are an E69K mutation, an L139P mutation, a D200N mutation, an E302R mutation, a T306K mutation, a W313F mutation, a T330P mutation, an L435G mutation, a P448A mutation, a D449G mutation, an N454K mutation, a D524N or D524A mutation, an L603W mutation, and an E607K mutation relative to SEQ ID NO: 3. 
     
     
         3 . The engineered reverse transcription enzyme of  claim 1 , wherein said engineered reverse transcription enzyme comprises: (i) three or more mutations selected from the group consisting of an L139 mutation, a D200 mutation, a T330 mutation, a P448 mutation, a D449 mutation, a D524 mutation, and a L603 mutation relative to SEQ ID NO: 3; and (ii) three or more mutations selected from the group consisting of an E69 mutation, an E302 mutation, a T306 mutation, a W313 mutation, an L435 mutation, and an N454 mutation relative to SEQ ID NO: 3. 
     
     
         4 . The engineered reverse transcription enzyme of  claim 3 , wherein said engineered reverse transcription enzyme comprises: (i) three or more mutations selected from the group consisting of an L139P mutation, a D200N mutation, a T330P mutation, a P448A mutation, a D449G mutation, a D524N or D524A mutation, and a L603W mutation relative to SEQ ID NO: 3; and (ii) three or more mutations selected from the group consisting of an E69K mutation, an E302R mutation, a T306K mutation, a W313F mutation, an L435G mutation, and an N454K mutation relative to SEQ ID NO: 3. 
     
     
         5 . The engineered reverse transcription enzyme of  claim 1 , wherein said engineered reverse transcription enzyme comprises: an E69 mutation, an L139 mutation, a D200 mutation, an E302 mutation, a T306 mutation, a W313 mutation, a T330 mutation, an L435 mutation, a P448 mutation, a D449 mutation, an N454 mutation, a D524 mutation, a D524 mutation, an L603 mutation, and an E607 mutation relative to SEQ ID NO: 3. 
     
     
         6 . The engineered reverse transcription enzyme of  claim 5 , wherein said engineered reverse transcription enzyme comprises: an E69K mutation, an L139P mutation, a D200N mutation, an E302R mutation, a T306K mutation, a W313F mutation, a T330P mutation, an L435G mutation, a P448A mutation, a D449G mutation, an N454K mutation, a D524N or D524A mutation, an L603W mutation, and an E607K mutation relative to SEQ ID NO: 3. 
     
     
         7 . The engineered reverse transcription enzyme of  claim 1 , wherein said truncation comprises a truncation of at least 20 amino acids from said N-terminus relative to SEQ ID NO: 3. 
     
     
         8 . The engineered reverse transcription enzyme of  claim 1 , wherein said truncation comprises a truncation of 23 amino acids from said N-terminus relative to SEQ ID NO: 3. 
     
     
         9 . The engineered reverse transcription enzyme of  claim 1 , wherein said engineered reverse transcription enzyme further comprises an affinity tag at said N-terminus or at a C-terminus of said amino acid sequence. 
     
     
         10 . The engineered reverse transcription enzyme of  claim 9 , wherein said affinity tag is at least 5 histidine amino acids. 
     
     
         11 . The engineered reverse transcription enzyme of  claim 9 , wherein said engineered reverse transcription enzyme further comprises a protease cleavage sequence, wherein cleavage of said protease cleavage sequence by a protease results in cleavage of said affinity tag from said engineered reverse transcription enzyme. 
     
     
         12 . The engineered reverse transcription enzyme of  claim 11 , wherein said protease cleavage sequence is a thrombin cleavage sequence. 
     
     
         13 . The engineered reverse transcription enzyme of  claim 12 , wherein said amino acid sequence comprises a MRSSHHHHHHSSGLVPRGS (SEQ ID NO: 7) amino acid sequence at said N-terminus. 
     
     
         14 . The engineered reverse transcription enzyme of  claim 11 , wherein said engineered reverse transcription enzyme is cleaved with said protease, thereby cleaving said affinity tag from said engineered reverse transcription enzyme. 
     
     
         15 . The engineered reverse transcription enzyme of  claim 1 , wherein said engineered reverse transcription enzyme comprises an amino acid sequence according to SEQ ID NO: 6. 
     
     
         16 . The engineered reverse transcription enzyme of  claim 15 , wherein said engineered reverse transcription enzyme comprises an amino acid sequence according to SEQ ID NO: 5. 
     
     
         17 . A method for nucleic acid sample processing, comprising: providing a template ribonucleic acid (RNA) molecule in a reaction volume and using the engineered reverse transcription enzyme of  claim 1  to reverse transcribe said RNA molecule to a complementary DNA molecule. 
     
     
         18 . The method of  claim 17 , wherein said reaction volume is less than 1 nanoliter. 
     
     
         19 . The method of  claim 18 , wherein said reaction volume is less than 500 picoliters. 
     
     
         20 . The method of  claim 17 , wherein said reaction volume is a droplet in an emulsion. 
     
     
         21 . The method of  claim 17 , wherein said reaction volume is a well. 
     
     
         22 . The method of  claim 17 , wherein said reaction volume further comprises a plurality of nucleic acid barcode molecules comprising a barcode sequence. 
     
     
         23 . The method of  claim 22 , wherein said RNA molecule is a messenger RNA (mRNA) molecule, wherein said plurality of nucleic acid barcode molecules further comprise an oligo(dT) sequence, and wherein said engineered reverse transcription enzyme reverse transcribes said mRNA molecule into said complementary DNA molecule using said oligo(dT) sequence, wherein said complementary DNA molecule comprises said barcode sequence. 
     
     
         24 . The method of  claim 22 , wherein said RNA molecule is a messenger RNA (mRNA) molecule, wherein said reaction volume further comprises a nucleic acid molecule comprising an oligo(dT) sequence, wherein said plurality of nucleic acid barcode molecules further comprise a template switching sequence, wherein said engineered reverse transcription enzyme reverse transcribes said mRNA molecule using said nucleic acid molecule comprising said oligo(dT) sequence, and wherein said engineered reverse transcription enzyme performs a template switching reaction, thereby generating said complementary DNA molecule, wherein said complementary DNA molecule comprises said barcode sequence. 
     
     
         25 . The method of  claim 22 , wherein said plurality of nucleic acid barcode molecules are attached to a support. 
     
     
         26 . The method of  claim 25 , wherein said nucleic acid barcode molecules are releasably attached to said support. 
     
     
         27 . The method of  claim 25 , wherein said support is a bead. 
     
     
         28 . The method of  claim 27 , wherein said bead is a gel bead. 
     
     
         29 . The method of  claim 17 , wherein said reaction volume comprises a cell comprising said RNA molecule. 
     
     
         30 . The method of  claim 29 , further comprising releasing said RNA molecule from said cell.

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