US2011143418A1PendingUtilityA1
Compositions and methods relating to orthogonal mrna pairs
Est. expiryJul 15, 2025(expired)· nominal 20-yr term from priority
C12N 15/67C12N 15/1034C12N 15/1089C12N 15/11
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Claims
Abstract
Orthogonal ribosome orthogonal mRNA pairs are provided, as are methods for their selection involving a novel positive-negative selection approach, and methods for their use. Also provided are cellular logic circuits involving orthogonal ribosomes.
Claims
exact text as granted — not AI-modified1 . An orthogonal mRNA orthogonal rRNA pair, wherein expression of said orthogonal rRNA is not toxic to the cell in which it is expressed.
2 . The orthogonal mRNA orthogonal rRNA pair of claim 1 , wherein said orthogonal mRNA comprises a sequence in the region from −13 to −7, relative to the AUG initiation codon, selected from the group consisting of: 5′, −13 CACCACX −7 -3′ (SEQ ID NO: 48, where X is a deletion of the nucleotide at −7 relative to the AUG); 5′- −13 CAACUGC −7 -3′ (SEQ ID NO: 49); 5′- −13 .CAUCCCU −7 -3′ (SEQ ID NO: 50); AND 5′- −13 UCCCUXX −7 -3′ ((SEQ ID NO: 51, where X is a deletion of the nucleotides at −7 and −8 relative to the AUG).
3 . The orthogonal mRNA orthogonal rRNA pair of claim 1 , wherein said orthogonal mRNA comprises a sequence immediately 5′ of the AUG initiation codon, selected from the group consisting of: 5′-CACCACCCGCAA-3′ (SEQ ID NO: 52); 5′-CAACUGCCCGCAA-3′ (SEQ ID NO: 53); 5′-CAUCCCUCCGCAA-3′ (SEQ ID NO: 54); and 5′-UCCCUCCGCAA-3′ (SEQ ID NO: 55).
4 . The orthogonal mRNA orthogonal rRNA pair of claim 1 , wherein said orthogonal rRNA comprises a sequence in the regions corresponding to nucleotides 722-723 and 1535-1540 of E. coli 16S rRNA selected from the group consisting of (a)-(j) below:
Orthogonal
rRNA sequence
722-723
1535-1540
a
AG
AGUGGU (SEQ ID NO: 56)
b
CG
CUGUGG (SEQ ID Na 57)
c
CA
UUGUGG (SEQ ID NO: 58)
d
GU
UUGUGG (SEQ ID NO: 59)
e
AC
AUGCAG (SEQ ID NO: 60)
f
AC
UUGCAG (SEQ ID NO: 61)
g
CG
UCGCAG (SEQ ID NO: 62)
h
CA
CCGCAG (SEQ ID NO: 63)
i
CA
UGGGAU (SEQ ID NO: 64)
j
GU
UGGGAU (SEQ ID NO: 65)
5 . The orthogonal mRNA orthogonal rRNA pair of claim 1 , wherein said orthogonal rRNA comprises a sequence in the regions corresponding to nucleotides 719-726 and 1531-1542 of E. coli 16S rRNA selected from the group consisting of (a)-(j) below:
Orthogonal
rRNA sequence
719-726
1531-1542
a
CCGAGUGGC
AUCAAGUGGUUA
(SEQ ID NO: 66)
(SEQ ID NO: 67)
b
CCGCGUGGC
AUCACUGUGGUA
(SEQ ID NO: 68)
(SEQ ID NO: 69)
c
CCGCAUGGC
AUCAUUGUGGUA
(SEQ ID NO: 70)
(SEQ ID NO: 71)
d
CCGGUUGGC
AUCAUUGUGGUA
(SEQ ID NO: 72)
(SEQ ID NO: 73)
e
CCGACUGGC
AUCAAUGCAGUA
(SEQ ID NO: 74)
(SEQ ID NO: 75)
f
CCGACUGGC
AUCAAUGCAGUA
(SEQ ID NO: 76)
(SEQ ID NO: 77)
g
CCGCGUGGC
AUCAUCGCAGUA
(SEQ ID NO: 78)
(SEQ ID NO: 79)
h
CCGCAUGGC
AUCACCGCAGUA
(SEQ ID NO: 80)
(SEQ ID NO: 81)
i
CCGCAUGGC
AUCAUGGGALTUA
(SEQ ID NO: 80)
(SEQ ID NO: 82)
j
CCGGUUGGC
AUCAUGGGAUUA
(SEQ ID NO: 83)
(SEQ ID NO: 82)
6 . The orthogonal mRNA orthogonal rRNA pair of claim 1 , wherein said pair comprises sequences selected from the paired mRNA and rRNA sequences:
Orthogonal mRNA:
−13 to −7
relative to AUG
orthogonal rRNA:
Pair
initiation codon
722-723/1535-1540
1
5′- −13 CACCACX −7 -3′
5′-A 722 G 72·3/1535 AGUGGU 1540 -3′
(SEQ 113 NO: 84)
(SEQ ID NO: 85)
2
5′- −13 CACCACX −7 -3′
5′ - C 722 G 723/1535 CUGUGG 1540 -3′
(SEQ ID NO: 84)
(SEQ ID NO: 86)
3
5′- −13 CACCACX −7 -3′
5′-C 722 A 723/1535 UUGUGG 1540 -3′
(SEQ ID NO: 84)
(SEQ ID NO: 87)
4
5′- −13 CACCACX −7 -3′
5′-G 722 U 723/1535 UUGUGG 1540 -3′
(SEQ ID NO: 84)
(SEQ ID NO: 87)
5
5′- −13 CAACUGC −7 -3′
5′-A 722 U 723/1535 AUGCAG 1540 -3′
(SEQ ID NO: 88)
(SEQ ID NO: 89)
6
5′- −13 CAACUGC −7 -3′
5′-A 722 C 723/1535 UUGCAG 1540 -3′
(SEQ ID NO: 88)
(SEQ ID NO: 90)
7
5′- −13 CAACUGC −7 -3′
5 ′- C 722 G7 23/1535 UCGCAG 1540 -3′
(SEQ ID NO: 88)
(SEQ ID NO: 91)
8
5′- −13 CAACUGC −7 -3′
5 ′- C 722 A 723/1535 CCGCAG 1540 -3′
(SEQ ID NO: 88)
(SEQ ID NO: 92)
9
5′- −13 CAUCCCU −7 -3′
5 ′- C 722 A 723/1535 UGGGAU 1540 -3′
(SEQ ID NO: 93)
(SEQ ID NO: 94)
10
5′- −13 CAUCCCU -7 -3′
5 ′- C 722 U 723/1535 UGGGAU 1540 -3′
(SEQ ID NO: 93)
(SEQ ID NO: 94)
7 . The orthogonal mRNA orthogonal rRNA pair of claim 1 , wherein said pair comprises mRNA and rRNA sequences selected from the pairs:
Orthogonal mRNA:
Orthogonal mRNA:
Immediately 5′ of AUG
719-726/1531-1542 relative
Pair
initiation codon
to E.coli 16S rRNA
a
5′-CACCACCCGCAA-3′
5′-719CCGAGGGC726 (SEQ ID NO: 96) /
(SEQ ID NO: 95)
1531 AUCAAGUGGUUA 1542 -3′ (SEQ ID NO: 97)
h
5′-CACCACCCGCAA-3′
5′-719CCGCGGGC726 (SEQ ID NO:98)/
(SEQ ID NO: 95)
1531 AUCACUGUGGUA l542 -3′ (SEQ ID NO: 99)
c
5′-CACCACCCGCAA-3′
5′-n9CCGCAGGC726 (SEQ ID NO: 100)/
(SEQ ID NO: 95)
1531 AUCAUUGUGGUA 1542 -3′ (SEQ ID NO: 101)
d
5′-CACCACCCGCAA-3′
5′-719CCGGUGGC (SEQ ID NO: 102)/
(SEQ ID NO: 95)
1531 AUCAUUGUGGUA 1542 -3′ (SEQ ID NO: 101)
e
5′-CAACUGCCCGCAA-3′
5′-719CCGACGGC726 (SEQ ID NO: 104)/
(SEQ ID NO: 103)
1531 AUCAAUGCAGUA 1542 -3′ (SEQ ID NO: 105)
f
5′-CAACUGCCCGCAA-3′
5′-n9CCGACGGC726 (SEQ ID NO: 104)/
(SEQ ID NO: 103)
1531 AUCALJUGCAGUA 1542 -3′ (SEQ ID NO: 106)
g
5′-CAACUGCCCGCAA-3′
5′-719CCGCGGGC726 (SEQ ID NO: 107/)
(SEQ ID NO: 103)
1531 AUCAUCGCAGUA 1542 -3′ (SEQ ID NO: 108)
h
5′-CAACUGCCCGCAA-3'
5′-719CCGCAGGC726 (SEQ ID NO: 109)/
(SEQ ID NO: 103)
1531 AUCACCGCAGUA 1542 -3′ (SEQ ID NO: 110)
5′-CAUCCCUCCGCAA-3′
5′-719CCGCAGGC726 (SEQ ID NO: 109)/
(SEQ ID NO: 111)
1531 AUCAUGGGAUUA 1542 -3′ (SEQ ID NO: 112)
j
5′-CAUCCCUCCGCAA-3′
5′-719CCGGUGGC726 (SEQ ID NO: 113)/
(SEQ ID NO: 111)
1531 AUCAUGGGAUUA 1542 -3′ (SEQ ID NO: 112)
indicates data missing or illegible when filed
8 . A fusion polypeptide comprising a positive selectable marker polypeptide and a negative selectable marker polypeptide, wherein expression of said fusion polypeptide permits cell survival in the presence of a positive selectable marker and renders cells sensitive to killing by said negative selectable marker.
9 . The fusion polypeptide of claim 8 , wherein said positive selectable marker comprises an antibiotic resistance coding sequence.
10 . The fusion polypeptide of claim 9 , wherein said antibiotic resistance coding sequence encodes chloramphenicol acetyltransferase.
11 . The fusion polypeptide of claim 8 , wherein said negative selectable marker comprises sequence encoding uracil phosphoribosyltransferase.
12 . The fusion polypeptide of claim 8 , wherein said positive selectable marker comprises chloramphenicol acetyltransferase and said negative selectable marker comprises uracil phosphoribosyltransferase.
13 . The fusion polypeptide of claim 8 , wherein éxpression of said fusion polypeptide in a cell permits said cell to survive in the presence of chloramphenicol and renders said cell sensitive to killing with 5-fluorouracil.
14 . The fusion polypeptide of claim 12 , wherein said uracil phosphoribosyltransferase is fused C-terminal to said chloramphenicol acetyltransferase.
15 . A vector comprising a sequence encoding a fusion polypeptide of claim 8 .
16 . The vector of claim 15 , wherein said sequence encoding a fusion polypeptide is operably linked to a sequence encoding a ribosome binding sequence.
17 . The vector of claim 16 , wherein said ribosome binding sequence is a mutated ribosome binding sequence.
18 . The vector of claim 17 , wherein said ribosome binding sequence comprises a mutation between −13 and +1 relative to an AUG initiation codon at the start of said sequence encoding a fusion polypeptide.
19 . A host cell comprising a vector of claim 15 .
20 . A cellular logic circuit comprising an orthogonal mRNA orthogonal ribosome pair.
21 . The cellular logic circuit of claim 20 , wherein said circuit comprises a Boolean AND circuit.
22 . The cellular logic circuit of claim 20 , wherein said circuit comprises a Boolean OR circuit.
23 . The cellular logic circuit of claim 20 , wherein said orthogonal mRNA orthogonal rRNA pair comprises a pair selected from those described in claim 6 .
24 . A cellular logic circuit or cascade comprising two or more orthogonal mRNA orthogonal rRNA pairs recited in claim 6 .
25 . A method of producing a polypeptide of interest, the method comprising: a) providing an orthogonal mRNA orthogonal rRNA pair, wherein said orthogonal mRNA comprises sequence encoding said polypeptide of interest; and b) introducing nucleic acid encoding said pair to a cell, wherein translation of said orthogonal mRNA by a ribosome comprising said orthogonal rRNA results in production of said polypeptide.
26 . The method of claim 25 , wherein said orthogonal mRNA orthogonal rRNA pair is a pair as described in claim 6 .Join the waitlist — get patent alerts
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