Table 5.

Selection for a low affinity binding population

DNA triplet	Sort	Amino acid at helix positions				Predicted net charge at pH 7.0
		Finger 1		Finger 2
		3	6	−1	2
N-AN(G)	4	G	A	R	D	0
		W	S	G	N	0
		W	I	R	D	0
		E	R	R	D	0
		L	R	R	D	0
N-CN(G)	4	H	D	R	S	0
		H	N	N	V	0
		H	H	R	D	0
		H	A	R	S	+1
N-TN(G)	4	H	R	R	S	+2
		H	E	R	S	0
		H	A	R	S	+1
		G	R	R	D	+1
		H	G	R	S	+1

Selection was for the 5′ base in the finger 1 binding site using a library containing substitutions on both finger 1 (positions 3 and 6) and finger 2 (positions −1 and 2). Selection for N-AN(G), N-CN(G), and N-TN(G) (where the fixed base occupies the 5′ position in the finger 1 binding site) results in a low affinity consensus class without binding enrichment. The upper part of the table shows selection for DNA triplet replacements of the finger 1 binding site using a library containing substitutions at positions −1, 2, 3, and 6 on the finger 1 recognition helix. The finger 1 binding site (GCG in GCG TGG GCG) was replaced by DNA sequences containing XNN, where X is a fixed base (A, C, G, or T) and N is any base. Binding enrichment only occurs for GNN, and results in complete consensus for Arg at position 6. The lower part of the table shows the selection for fixed middle bases. All clones in these high-affinity populations contain Arg at position 6 on the recognition helix, even though selection is for mixed bases at the 5′ position in the finger 1 binding site. Numbers in parentheses refer to multiple isolates of a particular clone.