66 Figure 2. GFP mRNA secondary structure
CNVK-AS (a-2), CNVK-AS (a-14) and CNVK-AS (a-6-G) were complementary to 14 nt~38 nt (Target Site A). CNVK-AS (a-2) and CNVK-AS (a-14) was including CNVK in the second-base position and the fourteenth-base positionfrom 5’, respectively. As a result of PAGE analysis, ORN-a and CNVK-AS (CNVK-AS (a-2) or CNVK-AS (a-14)) clearly disappeared and a band having mobility of about 40 nt appeared after UV irradiation (Fig. 3a,b). In addition, after the UV irradiation for 5 seconds, conversion of ORN-a did not confirm the remarkable difference in case of comparing CNVK-AS (a-2) with CNVK-AS (a-14) (Fig. 3e). On the other hand, these phenomena were not observed in the case of CNVK-AS (a-6-G) and CNVK-AS (a-2-invert) (Fig 3c,d).
CNVK-AS (a-6-G) was included CNVK in sixth-base positionfrom 5’. This CNVK-AS has no crosslinking ability to the complementary sequence because CNVK cannot crosslink to guanine. CNVK-AS (a-2-invert) is inverted control of CNVK-AS (a-2).
Output of sir_graph (©) mfold_util 4.6
Created Wed Sep 18 02:20:51 2013
dG = -243.25 [Initially -260.90] GFP mRNA
A U
G G G U A
G C A
A G G G C AG G A G G C U G U U C C A C
G G G G U G G G U C C C UA C C U G G U C G A G C U G G A C G G C
G A C G A U A A C
G G
C A C C
A A G
U U
C A
G C
G U
G U
C C
G G
CG A
G G
G C G A G
G G C A G GU CC CA C U AC GGCA
GA C U G A C C C U
G A A G U U C A U C U CG A C C A C C G G C A A G C U G C C C
G U G C C C U G G C C C A C C C U G C U G C A C A
C C C G U A C C A U C G
G C G U G C A G GU UC CU A CG GC C U A C C C
CG A C C A
C A
UGA A GCA CG
A C AG UC C U U
U C AA
GU CC
G C A C U G C C C GA A
G G
CU AC G U C C A G G A C G G C
AC C A
U C
U U
C U
U C A G A
G A
C G A C G G
C A A C UA C
A A
G A
C
C
C G
C G
C C
G A
G G
UGAA G U U C G A G G G C G A
C A C C
C U G G U G A
A C C G C A U C G A G C U G A A
G G
G C A U CG
A C U U
C A A
G G A
G G A
C G G
C AA C
A U CC U
G G
G G
C A
C A A G C
U G
G A
G U
A C A A
C U A C A A C A G C
CA C AA C
GU C
UA U
A U C A U G G C C G
A A C G A
C A
G A A
G A A C
G G C
A U
C A A
G G
U G
A A
C U
U C
A A
G A
U C C
G C
C A C A AC
A
U C G A G G A C G G C A G C G U G C A G C U C G C C G A C
CA C U A C C
A G
C A G A CA CA CC C C A C U G G G C A G C C G C C C UG G UC C G GU C C G C A C A A
C C
A C
U A C C
U G
A G
C A C
C C A G U
C C
G C
C C
UG A G
C AA A G A
C C C C
A A
C G
A G A A
G C
G C
G A
U C
A AC U G G U C C U G C GU AG
G U
U C
G U G
A C
C GC
C G
C C
G G
G A
U AC C U C U C G G C A
U G G A C G A G C G U AU AC A G U A A
5’
3’
40
80 120 160 200
240 280
320
360
400
440
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520
560 600
640
680 3’
Output of sir_graph (©) mfold_util 4.6
Created Wed Sep 18 02:20:51 2013
A U
G G G U A
G C A
A G G G C AG G A G G C U G U U C C A C
G G G G U G G G U C C C UA C C U G G U C G A G C U G G A C G G C
G C A G A U A A C
G G
C A C C
A A G
U U
C A
G C
G U
G U
C C
G G
CG A
G G
G C G A
G G G C A G GU CC CA C U AC GGCA
GA C U G A C C C U
G A A G U U C A U C U CG A C C A C C G G C A A
G C U G C C C
G U G C C C U G G C C C A C
C C U G C U G C A C A
C C C G U A C C AU C G G C G U G C A G GU UC CU A CG GC C U A C C C
CG A C C A
C A
UGA A GCA CG
A C G CA U C U U
U C A
AG U
C CG C A C U G C C C GA A
G G
CU AC G U C C A G G A C G G C
AC CA U C
U U
C U
U C A G A
G A
C G A C G G
C A A C UA C
A A
G A
C
C
C G
C G
C C
G A
G G
UG AA G U U C G A G G G C G A
C A C C
C U G G U G A
A CC G C A U C G A G C U G A A
G G
G C A U CG
A C U U
C A A
G G A
G G A
C G G
C AA C
A U C C U
G G
G G
C A
C A A G C
U G
G A
G U
A C A A
C U A C A A C A G C
CA C AA
CG UC
U AU
A U C A U G G C C G
A C A A G
C A
G A A
G A A C
G G C
A U
C A A
G G
U G
A A
C U
U C
A A
G A
U C C
G C
C A C A AC
A
U C G A G G A C G
G C A G C G U G C A G C U C G C C G A C
CA C U A C C
A G
C A G A A A C C C CC C A CU G G GC A C G C G CC G C G U U C G U C C G C C A G C A A
C C
A C
U A C C
U G
A G
C A CC
C A G U
C C
G C
C C
UG A G
C AA A G A
C C C C
A A
C G
A G A A
G C
G C
G A
U C
A AC U G G U C C U G C GU AG
G U
U C
G U G
A C
C G
C C G
C C CG G G C A
U G G A C G A G C G U AU C A A G U A A
5’
3’ 40
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240 280
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560 600
640
680 3’
Output of sir_graph (©) mfold_util 4.6
Created Wed Sep 18 02:20:51 2013
dG = -243.25 [Initially -260.90] GFP mRNA
A U
G G G U A
G C A
A G G G C AG G A G G C U G U U C C A C
G G G G U G G G U C C C UA C C U G G U C G A G C U G G A C G G C
G C A G AU A A C
G G
C A C C
A A G
U U
C A
G C
G U
G U
C C
G G
CG A
G G
G C G A
G G G C A G GU CC CA C U AC GGCA
GA C U GA C C C U
G A A G U U C A U C U CG A C C A C C G G C A A G
C U G C C C
G U G C C C U G G C C C A C
C C U G C U G C A C A
C C C G U A C C AU C G G C G U G C A G GU UC CU A CG GC C U A C C C
CG A C C A
C A
UGA A GCA CG
A C AG UC C U U
U C AA
G UCC
G C A C U G C C C GA A
G G
CU AC G U C C A G G A C G G C
AC C A
U C
U U
C U
U C A G A
G A
C G A C G G
C A A C UA C
A A
G A
C
C
C G
C G
C C
G A
G G
UG AA G U U C G A G G G C G A
C A C C
C U G G U G A
A C C G C A U C G A G C U G A A
G G
G C A U CG
A C U U
C A A
G G A
G G A
C G G
C AA C
A U CC U
G G
G G
C A
C A A G C
U G
G A
G U
A C A A
C U A C A A C A G C
CA C AA C
GU CU
A U
A U C A U G G C C G
A A C G A
C A
G A A
G A A C
G G C
A U
C A A
G G
U G
A A
C U
U C
A A
G A
U C C
G C
C A C A AC
A
U C G A G G A C G G C A G C G U G C A G C U C G C C G A C
CAC U A C C
A G
C A G A CA CA CC C C A CU G C G A G GC C G CC G C GU U C C G U C G C C G A C A A
C C
A C
U A C C
U G
A G
C A C
C C
A G U
C C
G C
C C
UG A G
C AA A G A
C C C C
A A
C G
A G A A
G C
G C
G A
U C
A AC U G G U C C U G C GU AG
G U
U C
G U G
A C
C GC
C G
C C
G G
G A
U AC C
U C U C G G C A
U G G A C G A G C G U A U C A A G U A A
5’
3’
40
80 120 160 200
240 280
320
360
400
440
480
520
560 600
640
680 3’
Output of sir_graph (©)
mfold_util 4.6 Created Wed Sep 18 02:20:51 2013
dG = -243.25 [Initially -260.90] GFP mRNA
AU G G GU A G C A A G G G C AG G AG G C U G U U C CA CGG G G U G G U G C C C UA C C U G G U C G A G C U G G A C G G C G A C G AU A ACG
G CC A CA A
GUU CAGC
GUGU CCGG
CGA GG
GC G A G G G C AG GU CC CA C U ACG
GCAA G C U GA CC CU
G A A G U U C A U C U CG A C CA C C G CG A AG C U G C C C
G U G C C C U G G C CC A C C C U GC U G A C C A C C C GU A C UC A C G G C UG G C A G GU UC CU A CG GC C U A C C CCG A C CA
C A UG AA
GC A CG A CAG C UU C UU C
AA GU
CCCG C A U G C CC GA A
GG CU
AC G U C C A G G A CG G CAC CA
U C U U C U U C
A GA
G A C G A C G G CA A C U
AC A A G A C C C
GC G C C G AG
GUGAA G U U C G A G G G C G A
C A C CC U
G G U GA A C C G C A U C G A G C U G A A G G G C A UCG
A C U U C AA
G G A G GA
C G G C AA C
A U C C U
G G G G C A
CA AGC UGGA
GUA C A A
C U A C A A C A G CCA C
AA CGUCU
AU A U AC U G G C C G A C A A G C A GAA
G A A C G GC
A U C A A G G U G A A C U U C
AA
G A U C C G C C A C A
AC A
U C G A G G A C GG C A G C G U G C A G CU C G C C G A
C CAC U A CC
A G C A G A A AC C CC C C A U GC G C G A GC G C C C GC U G C GU C U CG C C G A C A A C C A C U A C C U G A G C AC
CC A G U C C G C C C UG
A G C A
A A G A C
CCCA
A C G A G A A
G C G C G A U C A AC U G G U C C U G C GU AG G U U C G UG
A C C G C C G C C G G G A U AC C U C U C G G C A U G G A C G A G C U G AU C A A G U A A 5’
3’
40
80 120 160 200
240 280
320 360
400
440
480
520
560 600
640
680 3’
AS-a K-AS-a1 K-AS-a2 K-AS-a3
AS-b K-AS-b1 K-AS-b2
AS-c K-AS-c1
AS-d K-AS-d1 K-AS-d2 Target'
site'C
Target' site'A
Target' site'D
Target' site'B
Figure 3. photocrosslinking between CNVK-AS and its complementary ORNs. PAGE analysis of the mixture of ORN-a and CNVK-AS (a-2) (a) , CNVK-AS (a-14) (b), NVK-AS (a-2-invert) (c) or CNVK-AS (a-6) (d) after UV irradiation. (e) Time course of the remaining ORNs during the photocrosslinking reaction. Photoreactions were performed with a 1 : 1 mixture of CNVK-AS and ORN ([CNVK-AS] = [ORN] = 2 µM in PBS (-), 37 °C). Error bar = standard deviation.
This result shows that cnvK-ASs can photocrosslinke to complementary ORN sequence selectively at the single base level with a few seconds of UV irradiation.
Furthermore, we evaluated the photoreactivity other CNVK-AS that have different target sites. CNVK-AS (b-2), CNVK-AS (c-2), CNVK-AS (d-2) was complementary to 471 nt~495 nt (Target Site B), 592 nt~616 nt (Target Site C), 542 nt~566 nt (Target Site D). These CNVK-ASs also photocrosslinked to complementary ORNs.
e) a)
b) d)
20#nt 30#nt 40#nt 50#nt
0######0.1#####0.5######1#########5#######10######30
ORN###################.#########+########+#########+#########+########+########+########+########+
cnvK.AS#(a.2)#################+#########.#########+#########+#########+########+########+########+########+
60#nt
UV#Irradia?on#(sec) DNA##
Ladder#
20#nt 30#nt 40#nt 50#nt
0######0.1#####0.5######1#########5#######10######30
ORN###################.#########+########+#########+#########+########+########+########+########+
cnvK.AS#(a.6.G)##################+########.#########+#########+#########+########+########+########+########+
60#nt
UV#Irradia@on#(sec) DNA##
Ladder#
0"
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120"
140"
0" 5" 10" 15" 20" 25" 30" 35"
Amount'of'ORN'remained'/%
Irradia4on'4me'/'sec cnvK.AS"(a.2)""
cnvK.AS"(a.14)""
cnvK.AS"(a.2.invert)""
cnvK.AS"(a.6.G)"
20#nt 30#nt 40#nt 50#nt
0######0.1#####0.5######1#########5#######10######30
ORN###################.#########+########+#########+#########+########+########+########+########+
cnvK.AS#(a.2.invert)#################+#########.#########+#########+#########+########+########+########+########+
60#nt
UV#Irradia@on#(sec) DNA##
Ladder#
20#nt 30#nt 40#nt 50#nt
0######0.1#####0.5######1#########5#######10######30
ORN###################.#########+########+#########+#########+########+########+########+########+
cnvK.AS#(a.14)#################+#########.#########+#########+#########+########+########+########+########+
60#nt
UV#Irradia?on#(sec) DNA##
Ladder#
Photo-regulation of reverse-transcription of GFP mRNA in GFP-HeLa cells
A major difficulty with the phosphorothioate-antisense approach is that most phosphorothioate antisense oligonucleotides are ineffective against their targeted RNA because their low affinity for complementary RNA substantially limits their ability to invade RNA secondary structures. cnvK-AS’s target mRNA sites (Target Site A, B or C) form two types of structure. Target Site A and Target Site B form a long base-paired structure, whereas Target Site C consists of two internal loops (Fig.
2). To evaluate the photoreactivity of cnvK-ASs that have different target mRNA sites, it is necessary to quantitate the GFP-mRNA of cnvK-ASs treated cells. In this study, real-time reverse transcription polymerase chain reaction (qRT-PCR) protocol was adopted.
Total mRNAs were extracted from cells 2 hours after cnvK-ASs treatment with UV irradiation. They were transcribed by reverse transcriptase in the presence of oligo (dT) primer, followed by PCR reactions. If a photocrosslinking reaction occurred, the reverse transcription was completely inhibited by the steric hindrance caused by the formation of an irreversible photoadduct. As shown in Fig. 3a, 100 nM cnvK-AS (a-2) and cnvK-AS (a-14) caused 57 % and 42% inhibition of reverse transcription respectively with 10 seconds of UV irradiation. The inhibition rate of reverse transcription of cnvK-AS (a-2) containing cnvK in the second-base position from 5’
was 15% higher compared with cnvK-AS (a-14) containing cnvK in the fourteenth-base position from 5’. On the other hand, AS (a-con), cnvK-AS (a-6-G) and cnvK-AS (a-2-invert) did not inhibit reverse transcription regardless of whether receiving UV irradiation or not (Fig 4a). This result shows that cnvK-AS can photocrosslink to target mRNA site sequences selectively at the single base level with a few seconds of UV irradiation. Furthermore, we evaluated cnvK-AS (b-2) and
cnvK-AS (c-2) against different target sites of GFP mRNA. As a result, cnvK-AS (b-2) and cnvK-AS (c-2) were found to cause 47 % and 39% inhibition of reverse
transcription respectively with 10 seconds of UV irradiation. In addition, cnvK-AS (d-2) was found to cause 44 inhibition of reverse transcription respectively with 10 seconds of UV irradiation. cnvK-ASs targeting other regions of GFP mRNA (cnvK-AS (b-2, c-2, d-2)) show the same levels of gene silencing effect as cnvK-AS (a-2) (Fig 4b), although the secondary structure of these target regions would be different from each other (Fig. 2), suggesting the possibility that the K-AS invade the intramolecular duplex of the target region.
a)
b)
Figure 4. Down regulation of GFP expression in GFP-HeLa cells caused by photoirradiation with cnvK-ASs treatment.cnvK-ASs were 100 nM. Relative amount % was calculated from the CT values of the amplification curves of real-time PCR. The CT values were normalized with the CT values of the amplification curves of GAPDH in the same total RNA. Error bar = standard deviation (n = 3). The error bars indicate standard deviation of the mean. *P < 0.05.
0"
20"
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Control"
Control_U V+"
AS"(a6con)"
AS"(a6con)_U V+"
cnvK6AS"(a62)"
cnvK6AS"(a62)_UV+"
cnvK6AS"(a614)"
cnvK6AS"(a614)_U V+"
cnvK6AS"(a66)"
cnvK6AS"(a66)_UV+"
cnvK6AS"(a6invert)"
cnvK6AS"(a6invert)_UV+"
Rela%ve'amount'of'' GFP'mRNA''(%)'
:"UV%
:"UV+
0"
20"
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Control"
Control_UV+" 100nM"UV3"
100nM"UV+"
cnvK3AS"(a32)"
cnvK3AS"(a32)_UV+"
Rela>ve"amount"(%)"
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140"
Control"
Control_UV+" 100nM"UV3"
100nM"UV+"
cnvK3AS"(a32)"
cnvK3AS"(a32)_UV+"
Rela>ve"amount"(%)"
0"
20"
40"
60"
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Control"
Control_U V+"
AS"(a6con)"
AS"(a6con)_U V+"
cnvK6AS"(a62)"
cnvK6AS"(a62)_UV+"
AS"(b6con)"
AS"(b6con)_U V+"
cnvK6AS
"(b62)"
cnvK6AS"(b62)_UV+"
AS"(c6con)"
AS"(c6con)_U V+"
cnvK6AS
"(c62)"
cnvK6AS"(c62)_UV+"
AS"(d6con)""
AS"(d6con)"UV"+"
cnvK6AS
"(d62)"
cnvK6AS"(d62)UV"+"
Rela%ve'amount'of'' GFP'mRNA''(%)
This result shows that cnvK-ASs can bind to the target mRNA regardless of the different target site and difference in Tm value. The reason for this is thought to be because the cnvK-ASs and target site crosslink by forming a covalent bond between
cnvK and the target pyrimidine base. Therefore, it is easier to design the antisense sequence with cnvK-AS than with normal phosphorothioate-antisense. As shown in Fig. 5, the photo-regulation efficiency of cnvK-AS (a-2) has a dose dependent manner, and the 50% inhibition concentration (IC50) was 75 nM. The IC50
was 2- to 3-fold smaller than that of standard phosphorothioate antisense ODNs for endogenous GFP,6,11 suggesting that the irreversible covalent bond formation between K- AS ODNs and target mRNA enhances the antisense effect.
Figure 5. Dose dependency of cnvK-AS (a-2) for the down regulation of GFP gene expression. The error bars = standard deviation (n=3)
0"
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0" 20" 40" 60" 80" 100" 120" 140"
Rela%ve'amount'of' 'GFP'mRNA'(%)
[cnvK9AS'(a92)'/'nM]
UV irradiation timing dependent regulation of reverse-transcription of GFP mRNA in GFP-HeLa cells
Next, I examined the correlation between UV irradiation timing and the amount of intact GFP mRNA in cnvK-AS (a-2) transfected cells. In the experiment shown in Fig. 6a, media were changed to fresh media containing 0.5 % FBS after 5 hours transfection and cells were allowed to culture for another 2, 26, 50 hours. After that time total mRNAs were extracted from cells. To evaluate the amount of intact GFP mRNA, the total RNAs were subjected to qRT-PCR protocol. The result shows that remarkable inhibition of reverse transcription was not confirmed. By contrast, in the experiment shown in Fig. 6b, cells were washed with PBS buffer and UV irradiated for 10 seconds after 5 hours transfection. After 10 seconds UV irradiation, fresh media were applied and cells were allowed to culture for another 2, 26, 50 hours.
The result shows that 57 % inhibition of reverse transcription was confirmed after 2 hours of UV irradiation. Additionally, after 26 and 50 hours from 10 seconds of UV irradiation, 36%, 22% inhibition of reverse transcription was confirmed. This phenomena seems to be due to the constant production of mRNA by transfection from the gene. In the experiment shown in Fig 6c, cells were UV irradiated for 10 seconds after 24 hours transfection. The result shows that inhibition of reverse transcription was not confirmed before UV irradiation for 10 seconds, 58%
inhibition was confirmed after UV irradiation. In the experiment shown in Fig. 6d cells were UV irradiated for 10 seconds twice after 5 hours transfection and 24 hours incubation. The result shows that 87% inhibition of reverse transcription was confirmed after a second course of UV irradiation for 10 seconds. After 24 hours from the second course of UV irradiation for 10 seconds, 78% inhibition of reverse transcription was confirmed. These results showed that cnvK-AS (a-2) clearly causes inhibition of reverse transcription by UV irradiation in a time dependent fashion.
The ability to control gene expression by UV irradiation at desired timing would be useful in biotechnology, molecular biology and other applications.
Figure 6. Inhibition of reverse transcription of GFP mRNA by cnvK-AS (a-2) cnvK-AS (a-2) was 100 nM. Relative amount % was calculated from the CT values of the amplification curves of real-time PCR. The CT values were normalized with the CT values of the amplification curves of GAPDH in the same total RNA. Error bar = standard deviation (n = 3).
0 25 50 75 100 125 150
-10 0 10 20 30 40 50 60 0
25 50 75 100 125 150
-10 0 10 20 30 40 50 60
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Time after transfection / h Time after transfection / h
Time after transfection / h Time after transfection / h
Relative amount of GFP mRNA / %Relative amount of GFP mRNA / % Relative amount of GFP mRNA / %Relative amount of GFP mRNA / %
a)
c)
b)
d)
0 25 50 75 100 125 150
-10 0 10 20 30 40 50 60
0"
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Control"
Control_U V+"
AS"(a6con)"
AS"(a6con)_U V+"
cnvK6AS"(a62)"
cnvK6AS"(a62)_UV+"
AS"(b6con)"
AS"(b6con)_U V+"
cnvK6AS
"(b62)"
cnvK6AS"(b62)_UV+"
AS"(c6con)"
AS"(c6con)_U V+"
cnvK6AS
"(c62)"
cnvK6AS"(c62)_UV+"
AS"(d6con)""
AS"(d6con)"UV"+"
cnvK6AS
"(d62)"
cnvK6AS"(d62)UV"+"
Rela%ve'amount'of'' GFP'mRNA''(%)
0"
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Control"
Control_U V+"
AS"(a6con)"
AS"(a6con)_U V+"
cnvK6AS"(a62)"
cnvK6AS"(a62)_UV+"
AS"(b6con)"
AS"(b6con)_U V+"
cnvK6AS
"(b62)"
cnvK6AS"(b62)_UV+"
AS"(c6con)"
AS"(c6con)_U V+"
cnvK6AS
"(c62)"
cnvK6AS"(c62)_UV+"
AS"(d6con)""
AS"(d6con)"UV"+"
cnvK6AS
"(d62)"
cnvK6AS"(d62)UV"+"
Rela%ve'amount'of'' GFP'mRNA''(%)
0"
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Control"
Control_U V+"
AS"(a6con)"
AS"(a6con)_U V+"
cnvK6AS"(a62)"
cnvK6AS"(a62)_UV+"
AS"(b6con)"
AS"(b6con)_U V+"
cnvK6AS
"(b62)"
cnvK6AS"(b62)_UV+"
AS"(c6con)"
AS"(c6con)_U V+"
cnvK6AS
"(c62)"
cnvK6AS"(c62)_UV+"
AS"(d6con)"" AS"(d6con)"UV"+"
cnvK6AS
"(d62)"
cnvK6AS"(d62)UV"+"
Rela%ve'amount'of'' GFP'mRNA''(%)
0"
20"
40"
60"
80"
100"
120"
140"
160"
Control"
Control_U V+"
AS"(a6con)"
AS"(a6con)_U V+"
cnvK6AS"(a62)"
cnvK6AS"(a62)_UV+"
AS"(b6con)"
AS"(b6con)_U V+"
cnvK6AS
"(b62)"
cnvK6AS"(b62)_UV+"
AS"(c6con)"
AS"(c6con)_U V+"
cnvK6AS
"(c62)"
cnvK6AS"(c62)_UV+"
AS"(d6con)"" AS"(d6con)"UV"+"
cnvK6AS
"(d62)"
cnvK6AS"(d62)UV"+"
Rela%ve'amount'of'' GFP'mRNA''(%)
Photo-regulation of GFP expression in GFP-HeLa cells
To determine the efficacy of cnvK-ASs at regulating GFP knockdown by UV irradiation, GFP-HeLa cells were transfected with cnvK-AS (a-2). Cells in one group of experiments were washed with PBS buffer and UV irradiated after 5 hours transfection. After 10 seconds of UV irradiation, fresh media were applied and cells were allowed to culture for another 24 hours. In the other group, cells were treated in the same way but with no UV irradiation. The GFP signals of the cells were examined by confocal laser scanning microscopy. All images were taken under the identical imaging conditions for all experimental groups. Fig. 7 shows the result of photoregulation of GFP expression with cnvK-AS (a-2). cnvK-AS (a-2) inhibited GFP translation only in the case of UV irradiation for 10 seconds. This loss of GFP is not the result of UV quenching, since no transfected cells not conformed loss of GFP after UV irradiation (Fig. 7f).
Figure 7. Photomodulation of GFP expression in GFP-HeLa cells.
In a 96-well plate, GFP-HeLa cells were transfected with cnvK-AS (a-2) using Lipofectamine ® RNAiMAX Reagent. (a,e) GFP-HeLa cells . (b,f) GFP-HeLa cells were UV irradiation [366 nm (1600 mW/cm2), 10 sec].(c,g) GFP-Hela Cells transfected with cnvK-AS-ODN (GFP) . (d,h) GFP-Hela Cells transfected with cnvK-AS-ODN (GFP) were UV irradiation [366 nm (1600 mW/cm2), 10 sec].
control
control
a) b) control c) cnvK,AS,ODN2(a,2) d)cnvK,AS,ODN2(a,2)
UV+ UV+
UV+ UV+
e) f) control g) cnvK,AS,ODN2(a,2) h) cnvK,AS,ODN2(a,2)
As shown in Fig. 8, the fluorescence intensity of GFP in cells was clearly decreased 40% by the photoirradiation with the treatment of K-AS-a1, suggesting that the K-AS-a1 can down regulate the cellular synthesis of GFP by 10 sec of photoirradiation.
Figure 8. Fluorescence intensity of GFP in cells before and after the treatment of antisense (K-AS-a1) and 10 sec of 366 nm irradiation. The error bars indicate the standard error of the mean. * P < 0.05.
0"
20"
40"
60"
80"
100"
120"
control"
control_U V+"
cnvK4AS"(a42)""
cnvK4AS"(a42)_UV+"
Rela%ve'fluoresoence'' intensity'of'GFP'(%)
Comparison of lipofection reagent
I compared the antisense effects of cnvK-AS (a-2) transfected in GFP-HeLa cells by Lipofectamine 2000 transfection reagent (Invitrogen) with antisense effects of
cnvK-AS (a-2) transfected in GFP-HeLa cells by Lipofectamine RNAiMAX transfection reagent (Invitrogen). As shown in Fig. 9, in the case of cnvK-AS (a-2) transfected by Lipofectamine 2000 transfection reagent, significant down regulation of GFP mRNA was induced by 366 nm photoirradiation as well as in the case of cnvK-AS (a-2) transfected by Lipofectamine RNAiMAX.
Figure 9. Comparison of lipofection reagent.cnvK-ASs were 100 nM. Relative amount % was calculated from the CT values of the amplification curves of real-time PCR. The CT values were normalized with the CT values of the amplification curves of GAPDH in the same total RNA. GFP mRNA wasquantified by real-time RT-PCR at 2 h after the photoirradiation. Error bar = standard deviation (n = 3). The error bars indicate standard deviation of the mean. *P <
0.05.
:"UV%
0"
20"
40"
60"
80"
100"
120"
140"
Control"
Control_UV+"
100nM"UV3"
100nM"UV+"
cnvK3AS"(a32)"
cnvK3AS"(a32)_UV+"
Rela>ve"amount"of" "GFP"mRNA"(%)"
0"
20"
40"
60"
80"
100"
120"
140"
Control"
Control_UV+" 100nM"UV3"
100nM"UV+"
cnvK3AS"(a32)"
cnvK3AS"(a32)_UV+"
Rela>ve"amount"(%)"
:"UV+
0"
20"
40"
60"
80"
100"
120"
140"
Control"
Control_UV+" 100nM"UV3"
100nM"UV+"
cnvK3AS"(a32)"
cnvK3AS"(a32)_UV+"
Rela>ve"amount"(%)"
cnvK%AS"(a%2) Lipofectamine""
2000 Lipofectamine""
RNAiMax