Anti-bovine Prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its β isoform with high affinity

Figures & data

Figure 1 Binding of apt #1 to bPrP and bPrP-β. Binding curves of apt #1 to bPrP and bPrP-β are shown by closed circles and open circles, respectively. Solid and dashed lines represent different buffer conditions: 10 mM K⁺ and 100 mM Na⁺, respectively. The binding data are analyzed by GraphPad PRISM (see Materials and Methods).

Figure 2 Detection of bPrP^C in bovine brain homogenate and dot-blotting assay using a biotinylated apt #1 (Bi#1). (A) Bovine brain homogenate was analyzed using SDS-PAGE and subsequently with Northwestern blotting. bPrP^C (lane 1 and 2) was detected using Bi#1 (left) and T2 antibody (right). Lane M represents protein molecular weight markers. N, M and D indicate non-, mono- and di-glycosilated PrP isoforms, respectively. (B) Dot-blotting assay of recombinant bPrP using Bi#1 or BiC#1. Number 1 represents cross-linked 5′-biotinylated RNA (Bi#1, BiC#1). Numbers 2–6 represent decreasing concentration of mounted bPrP (ng) as follows: 2 = 480, 3 = 240, 4 = 120, 5 = 60, 6 = 30.

Figure 3 CD spectra of apt #1 and r(GGA)₄-15 in the presence of KCl. CD spectra were measured at 20°C in titration with KCl (0, 1, 10, 100 mM). (A) CD spectra of 10 µM apt #1. (B) The structure of d(GGA)₄.24

Figure 4 Binding affinity of anti-bPrP aptamer apt #1 for deletion variants of bPrP. (A) Circles, triangles and squares indicate full length bPrP (25–241), bPrP (102–241) and bPrP (132–241), respectively. (B) The amino-acid sequence alignment of bPrP. The apt #1 binding region is underlined.

Figure 5 Comparison of binding of apt #1 and bPrP using competitive binding assay. The #1 mutants (m7, m8) contained substitutions (shown underlined) in the conserved region (GGA)₄ as follows: m7: 5′-GGUGGAGGAGGA-3′; m8: 5′-GGUGGUGGUGGU-3′. The competitor RNAs were added in various concentrations with 0, 1, 5 and 10-fold greater molar concentrations of labeled apt #1. Binding assay was performed in the presence of 10 mM KCl (A) and in the absence of KCl (B).

Table 1 In vitro selection conditions for recombinant bPrP

Round of selection	RNA (µM)	bPrP (µM)	tRNA (µM)	Anti-PrP RNA aptamer16 (µM)	Time (min)
1	10	2	0	0	60
2	6	0.4	10	0	30
3	4	0.2	40	8	20
4	3	0.1	100	16	15
5	2	0.3	100	32	10
6	2	0.05	100	48	10
7	2	0.05	*6	0	10
8**	2	0.05	100	64	10
9**	2	0.05	100	64	10
10	6	0.05	100	96	10

* U₁₃ was used instead of tRNA.

** The mutagenic PCR was introduced.

Table 2 Sequences of randomized regions and majorities of isolated RNA aptamers against recombinant bPrP

Name of clone	Number of clones	Sequence of randomized region
#1	14/30	5′-CAAUCCAUUCAUCUCUCGAAUGAGGAAGUAGCCCAAGAGGAGGAGGAGGAUGAGC-3′
#6	11/30	5′-ACCUUCUGUUCAUAUCGUGACCAACCCAAUAGAUUGUGAUAAAGGAGGAGGAGGA-3′
#20	3/30	5′-UUGCCAAUAGCCCUACGGAGGAGGAGGCUGGACUAUAGACAGUUUACUUAAUAAA-3′
others		5′-CGCUCUGCCUGCGACCCAUCGACGUGAGUUUGCGAAAAAAGGAGGAGGAGGA-3′
		5′-UAGCCCCCCUAGGAGCCGCUUGUGAUAAGAGUUCAAAUUGGAGGAGGUGGAUUGA-3′

The conserved GGA triplet repeats (GGA)₄ are underlined involving its one point mutations.

Table 3 Sequences and binding affinities of different mutants

aptamers	sequence	Kd (nM)
		bPrP	bPrP-β
apt #1	5′-GGAGGAGGAGGA-3′	31 ± 6	220 ± 67
m1	5′-GGAGGAGGA-3′	79 ± 24	≥1000
m2	5′-GGAGGA-3′	75 ± 28	≥1000
m3	5′-(no GGA repeat)-3′	55 ± 24	≥1000
m4	5′-UUUUUUUUUUUU-3′	134 ± 42	≥1000
m5	5′-UUAUUAUUAUUA-3′	56 ± 16	≥1000

Table 4 Comparison of binding affinities for bPrP and bPrP-β between GGA repeat containing RNAs and DNA

RNA/DNA	sequence	Kd (nM)
		bPrP	bPrP-β
apt #1	-	31 ± 6	220 ± 70
r(GGA)4	r(GGAGGAGGAGGA)	8.5 ± 3.4	280 ± 80
d(GGA)4	d(GGAGGAGGAGGA)	64 ± 31	≥1000
R14	r(GGAGGUUUUGGAGG)	32 ± 18	610 ± 240

Table 5 Comparison of adenine stretch at 5′-site of (GGA)₄ with binding affinities of bPrP, bPrP(102–241) and bPrP-β

aptamers	sequence		Kd (nM)
		bPrP	bPrP (102–241)	bPrP-β
r(GGA)4-19	AAAAAAAGGAGGAGGAGGA	17 ± 5	540 ± 310	270 ± 90
r(GGA)4-18	AAAAAAGGAGGAGGAGGA	10 ± 2	160 ± 50	150 ± 40
r(GGA)4-17	AAAAAGGAGGAGGAGGA	9.4 ± 4.0	270 ± 120	78 ± 30
r(GGA)4-16	AAAAGGAGGAGGAGGA	16 ± 3	190 ± 50	92 ± 26
r(GGA)4-15	AAA GGAGGAGGAGGA	16 ± 4	220 ± 90	200 ± 90
r(GGA)4	GGAGGAGGAGGA	8.5 ± 3.4	240 ± 90	280 ± 80

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