Modified ARCA analogs providing enhanced translational properties of capped mRNAs

Figures & data

Figure 1. Structure and synthesis of new cap analogs. (i) MeCN, DMAP, Et₃N, Ac₂O, MeOH; (ii) NPE-OH, PPh₃, DIAD, toluene, (iii) HF/pyridine, tBuONO, pyridine; (iv) RNH₂, DMSO; (CH₃)₂NH/EtOH; (v) POCl₃, trimethyl phosphate; (vi) MeI, DMSO; (vii) imGDP, ZnCl₂, DMF

Table 1. Inhibition of capped luciferase mRNA translation in RRL lysate by N2-modified cap analogs. Data represent the means ± SD of at least three independent repeats. Statistical significance between m⁷GpppG and N2-modified cap analogs was determined using ANOVA and Tukey’s multiple comparison tests using GraphPad Prism 6

cap analog	compound #	IC50 [µM]	P value
m^7GpppG		11.56 ± 3.2
bn^2m2^7,3ʹOGpppG	9	4.46 ± 0.53	< 0.05
(p-OCH3bn)^2m2^7,3ʹOGpppG	10	4.12 ± 0.35	< 0.05
bn^2m2^7,2ʹOGpppG	11	1.7 ± 0.16	< 0.05

Table 2. Translational properties of differently capped mRNA in RRL and HEK293 cells. Data represent the means ± SD of at least three independent repeats

	Rabbit Reticulocyte Lysate		HEK293 cells
cap analog	relative translation efficiency	relative cap-dependent translation efficiency	relative total protein expression
m^7GpppG	1	1	1
ApppG	0.11 ± 0.03	0	0.52 ± 0.30
m2^7,2ʹ-OGpppG	1.59 ± 0.18	1.67 ± 0.20	nd
m2^7,3ʹ-OGpppG	1.56 ± 0.03	1.65 ± 0.06	1.46 ± 0.50
bn^2m2^7,3ʹ-OGpppG (9)	1.39 ± 0.19	1.44 ± 0.22	3.30 ± 0.86
(p-OCH3bn)^2m2^7,3ʹ-OGpppG (10)	1.29 ± 0.17	1.35 ± 0.20	2.42 ± 0.30
bn^2m2^7,2ʹ-OGpppG (11)	1.72 ± 0.28	1.82 ± 0.34	2.87 ± 0.50

Figure 2. Translation efficiency of analog-capped mRNAs in HEK293 cells. (a) Luciferase activity measured in HEK293 cells transfected with mRNAs capped with following analogs: bn²m₂^7,3ʹOGpppG, bn²m₂^7,2ʹOGpppG, (p-OCH₃bn)²m₂^7,3ʹOGpppG, m₂^7,3ʹ-OGpppG, m⁷GpppG. Luciferase activity was normalized to the protein concentration of samples and then expressed relatively to controls. The graph shows the mean of 4 independent experiments with ± SD. (b) Immunofluorescence staining of luciferase protein. Expression level of luciferase was detected after 24 h of mRNA transfection of HEK293 in use of anti-luciferase antibodies followed by anti-mouse FITC antibody (green signal). Cell nuclei were visualized with DAPI (blue color)

Figure 3. Stability of analog-capped mRNAs. (a) SpDcp1/2 hydrolysis of capped 34nt-RNAs. The transcripts were synthesized with T7 polymerase in use of an EcoRI digested pSP-luc+ template. Samples were resolved on 16% denaturing polyacrylamide/urea gel. Stained with SYBR gold. (b) Effect of the 5ʹ cap analogs on half–life of mRNAs in HEK293 after lipofection. RNA was isolated from cells 4, 24 and 72 h after transfection. Luciferase transcripts levels were quantified by qRT-PCR. ACTB gene was used as a reference gene. Data represent the means ± SD of three independent repeats

Table 3. Susceptibility of differently capped mRNAs to decapping by SpDcp1/Dcp2 complex

	Decapping %
cap analog	5 min	30 min
m2^7,3ʹ-OGpppG	13.7 ± 20.0	48.2 ± 7.5
bn^2m2^7,3ʹ-OGpppG (9)	53.4 ± 20.0	62.5 ± 1.15
(p-OCH3bn)^2m2^7,3ʹ-OGpppG (10)	35.4 ± 10.8	46.3 ± 18.6
bn^2m2^7,2ʹ-OGpppG (11)	65.8 ± 7.0	72.7 ± 4.60

Table 4. Stability of differently capped mRNAs in HEK293 cells

	Remaining mRNAs in %
cap analog on 5ʹ end	4h	24h	48h
m^7GpppG	100	67.4 ± 1.4	36.8 ± 2.6
ApppG	100	48.5 ± 0.5	37.0 ± 1.5
m2^7,3ʹ-OGpppG	100	71.7 ± 1.0	54.1 ± 1.5
bn^2m2^7,3ʹOGpppG (9)	100	95.0 ± 3.1	76.7 ± 1.0
(p-OCH3bn)^2m2^7,3ʹOGpppG(10)	100	73.3 ± 2.0	55.1 ± 1.6
bn^2m2^7,2ʹOGpppG (11)	100	85.7 ± 3.0	76.5 ± 4.6

Table 5. Binding affinities of N2-modified cap analogs to eIF4E derived from fluorescence titrations in HEPES/KOH 50 mM pH 7.2, KCl 200 mM, DTT 1 mM, EDTA 0.5 mM, at 20°C

cap analog	compound #	Kd [nM]
m2^7,2ʹ-OGpppG		100.5 ± 6.4
bn^2m2^7,3ʹOGpppG	9	182 ± 13
(p-OCH3bn)^2m2^7,3ʹOGpppG	10	880 ± 170
bn^2m2^7,2ʹOGpppG	11	30.5 ± 1.7

Figure 4. Increased affinity to eIF4E of N2-modified cap analogs. (a) Binding curves obtained by intrinsic protein fluorescence quenching upon the complex formation with (◆) bn²m₂^7,2ʹOGpppG, (○) m₂^7,2ʹ-OGpppG, (■) bn²m₂^7,3ʹOGpppG, and (▼) (p-OCH₃bn)²m₂^7,3ʹOGpppG. (b) Correlation between the affinity to eIF4E and the relative cap-dependent translation efficiency