Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA

Table 1 Mutations, cognate and noncognate RNA oligonucleotide sequences, and binding affinities of PUF (WT), and PUFs S2-S8

Protein	Protein modules	Cognate RNA sequence	K_D, nM	Noncognate RNA sequence	K_D, nM
	Ct 8 – 7 – 6 – 5 – 4 – 3 – 2 – 1 Nt	5′ 1 2 3 4 5 6 7 8 3′		5′ 1 2 3 4 5 6 7 8 3′
PUF(WT)	NQ-SE-NQ-CQ-NQ-CQ-NQ-SQ	CCAGAAU/UGUAUAUA/UUCG	0.043±0.023	CCAGAAU/A GUAUAUU/ AUUCG	3.56±0.90
PUF (S2)	CQ-SE-NQ-CQ-NQ-CQ-NQ-NQ	CCAGAAU/A GUAUAUU/ AUUCG	0.76±0.11	CCAGAAU/UGUAUAUA/UUCG	6.29±2.80
PUF (S4)	NQ-SE-CQ-NQ-CYQ-NQ-NQ-SQ	CCAGAAU/UGAUAU UA/UUCG	0.59±0.10	CCAGAAU/UGUAUAUA/UUCG	5.77±1.33
PUF (S6)	CQ-SE-CQ-NQ-CYQ-NQ-NQ-NQ	CCAGAAU/A GAUAU UU/ UUCG	6.05±0.25	CCAGAAU/UGUAUAUA/UUCG	89.6±16.5
PUF (S8)	CQ-NQ-CQ-NQ-CYQ-NQ-SE-NQ	CCAGAAU/AUAUAUGU/ UUCG	2.79±0.69	CCAGAAU/UGUAUAUA/UUCG	ND

Bold, mutant modules and changed RNA bases compared with the WT target sequence. Target RNA sequences are delimitated by forward slashes. Ct, C-terminus, and Nt, N-terminus of the protein. K_D values were determined using nonlinear curve fitting and represent the mean ± SD (n = 3). ND, K_D not determined due to little binding at 300 nM PUF (S8) to WT RNA.

ISSN: 1754-1611