Short loop-targeting oligoribonucleotides antagonize Lin28 and enable pre-let-7
processing and suppression of cell growth in let-7 deficient cancer cells.
Martina Roos1, Mario A. E. Rebhan1, Matije Lucic1, David Pavlicek1, Ugo Pradere1, Harry
Towbin1, Gianluca Civenni2, Carlo V. Catapano2, Jonathan Hall1*
1
Supplementary Materials
Tables and Figures
Page
Table S1: Sequences of oligonucleotides, masses calculated and masses observed
3
Figure S1: Looptomirs assayed for binding to truncated biotinylated pre-let-7a-2 bound to the
4
surface of a streptavidin-coated biosensor by surface plasmon resonance (SPR) spectroscopy
Figure S2: Inhibition by looptomirs of Lin28A binding to immobilized pre-let-7a-2.
4
Figure S3: Looptomirs inhibit Lin28A binding (upper panel) but not hnRNPA1 to pre-let-7a-2.
5
Figure S4: Cellular localization of Cy5-labelled L29-13 in Huh7 cells.
5
Figure S5: Detection of endogenous Lin28B by immunofluorescence in HepG2 and Huh7 cells
6
Figure S6: Levels of mature let-7a 48 h after transfections of L29-13, L30-14 and Lcon
6
Figure S7 Uncropped Western blot membranes showing Lin28B and B-Actin in HepG2 lysates
7
Figure S8: Western blot showing levels of let-7 target Lin28B after treatment of HepG2 cells
7
Figure S9 Secondary structures of pre-miR-18a, pre-mir-122 and pre-let-7a-2.
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2
Table S1: Sequences of oligonucleotides used in the study, masses calculated and masses observed.
Oligoribonucleotide
name
Sequence 5’ -> 3’
Type
Mass
calc.[g/mol]
Mass obs
[g/mol]
Let-7a-2 total
complementary + primer
+ restriction sites
CCTCCACTTCAGCCAGGACTCGAGTTTTCATTTTGAAG
GGGCCTCACCGAGTGGGGGCATCATCAAAAACTTTAA
CTATACAACCTACTACCTCAGGAGTCCCCTCACCTCCT
CTAAGGTTGGGCAGGGTGACCCTGAAGTGAGCACAG
CCTAGGGCTGAGCTGGGGACCTGGTGCGGCCGCTGA
GTCTTCGGACCTCGC
DNA
-
-
Let-7a-2 total
complementary mutated
+ primer + restriction
sites
CCTCCACTTCAGCCAGGACTCGAGTTTTCATTTTGAAG
GGGCCTCACCGAGTGGGGGCATCATCAAAAACTTTAA
CTATACAACCTACGAACGCAGGAGTCCCCTCACCTCC
TCTAAGGTTGGGCAGGGTGACCCTGAAGTGAGCACA
GCCTAGGGCTGAGCTGGGGACCTGGTGCGGCCGCTG
AGTCTTCGGACCTCGC
DNA
-
-
Pre-let-7a-2 T7 DNA
template
GGAAAGCTAGGAGGCTGTACAGTTATCTCCCTTGATG
TAATTCTAAACTATACAACCTACTACCTCCTATAGTGA
GTCGTATTAGGATCC
DNA
-
-
T7 DNA Polymerase
Promotor
GGATCCTAATACGACTCACTATAG
DNA
-
-
siRNA lin28b5’
AAAUCCUUCCAUGAAUAGUTT
RNA/DNA
6852
-
siRNA lin28b3’
ACUAUUCAUGGAAGGAUUUTT
RNA/DNA
6909
-
siRen 5’
GAGCGAAGAGGGCGAGAAAUU
RNA
6901.31
6902.5
siRen 3’
UUUCUCGCCCUCUUCGCUCUU
RNA
6436.78
6436.2
Pre-let-7a-2
UGAGGUAGUAGGUUGUAUAGUUUAGAAUUACAUCAA
GGGAGAUAACUGUACAGCCUCCUAGCUUUCC
RNA
21'502.9
21'503.3
Pre-mir-18a + Biotin(X)
UAAGGUGCAUCUAGUGCAGAUAGUGAAGUAGAUUAG
CAUCUACUGCCCUAAGUGCUCCUUCUGGX
RNA
-
-
Pre-mir-122 + Biotin (X)
UGGAGUGUGACAAUGGUGUUUGUGUCUAAACUAUCA
AACGCCAUUAUCACACUAAAUAX
RNA
-
-
Pre-let-7a-2 truncated +
linker + Biotin(X)
AGGUUGUAUAGUUUAGAAUUACAUCAAGGGAGAUAA
CUGUACAGCCUCTTTTTTUX
RNA/DNA
18'039.3
18'039.6
Lcon
AUGUAGUUCCCUC
2’-OMe RNA
4'220.8
4'220.1
L35-09
AUCUCCCUU
2’-OMe RNA
2'838.9
2'838.3
L33-11
AUCUCCCUUGA
2’-OMe RNA
3'541.4
3'540.8
L31-13
AUCUCCCUUGAUG
2’-OMe RNA
4'220.8
4'220.1
L31-09
CCCUUGAUG
2’-OMe RNA
2'918.0
2'917.2
L30-14
AUCUCCCUUGAUGU
2’-OMe RNA
4'541.0
4'540.3
L30-13
UCUCCCUUGAUGU
2’-OMe RNA
4'197.8
4'197.1
L29-13
CUCCCUUGAUGUA
2’-OMe RNA
4'220.8
4'220.2
L29-13 + Cy5-label
CUCCCUUGAUGUA
2’-OMe RNA
4833.61
4832.8
L29-09
CUUGAUGUA
2’-OMe RNA
2'943.0
2'942.4
General primer 1
forward
CCTCCACTTCAGCCAGGA
DNA
-
-
General primer 1 revers
GCGAGGTCCGAAGACTCA
DNA
-
-
3
a
for binding to tru
uncated biotinylated pre--let-7a-2 bou nd to the surrface of a
Fig. S1: Looptomirs assayed
streptaviidin-coated biosensor
b
by surface plassmon resona
ance (SPR) spectroscopy
s
y.
Fig. S2: Inhibition by
b looptomirs
s of Lin28A binding to immobilized
i
pre-let-7a-2 (see Fig. 1 in main
nt).
documen
4
Fig. S3 Binding curvves of loopto
omirs inhibiti ng Lin28 bin
nding (upperr panel) but not hnRNPA
A1 (lower
panel) to
o pre-let-7a-2
2. The bindin
ng curves of tthe two prote
eins were assed in paralllel.
Figure S
S4: Cellular localization
l
of
o looptomirss. Huh7 cells were trans
sfected with Cy5-labelled
d L29-13
(Table S
S1) at 20 nM concentratio
ons under sta
ditions (See Methods).
M
L229-13 accum
mulates in
andard cond
the nucle
eus and the cytoplasm 12 h post tran
nsfection as shown by liv
ve fluorescennce images of
o L29-13
(in green). Scale ba
ar indicate 15 μm; pictu
ocessing details are givven in Mate
erials and
ure post pro
Methodss.
5
Figure S
S5: Detection
n of endogen
nous Lin28B
B (in green) by immunofluorescence in HepG2 and
a Huh7
cells; sca
ale bars indiccate 15 μm. Picture post processing details are given in Mateerials and Me
ethods.
Figure S
S6: Endogeno
ous levels off mature let-7
7a 48 h afterr transfection
ns of L29-13,, L30-14 and
d Lcon into
liver Hep
pG2 cells, me
easured by TaqMan
T
RT- qPCR (norm
malized to Lcoon).
6
Figure S
S7 Uncroppe
ed Western blot
b membran
nes showing
g Lin28B and
d B-Actin dettection in He
epG2 cell
lysate an
nd Lin28B, HMGA2
H
and B-Actin
B
detecction in Huh7
7 cell lysate.
Figure S
S8: Western blot showing
g levels of le
et-7 target protein
p
Lin28B after treattment of Hep
pG2 cells
with loop
ptomirs. Uncropped pictu
ures are show
wn in Fig. S7
7
7
o pre-miR-1
18a, pre-mir--122 and pre-let-7a-2 ass predicted by Mfold
Fig. S9: Secondary structures of
3; Zuker and Jacobson, 1998)).
1
(Waugh et al., 2002; Zuker, 2003
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