Oligonucleotides Unmodified DNA oligonucleotides were purchased

Oligonucleotides Unmodified DNA oligonucleotides were purchased from Hokkaido System Science (Hokkaido, Japan) and Fasmac (Tokyo, Japan). 5´-Carboxyfluorescein (FAM)-labeled DNA/RNA chimeric oligonucleotide and IRD700-labeled DNA oligonucleotide were purchased from IDT (Coralville, IA) and LI-COR (Lincoln, NE), respectively. 5´-Biotin-modified RNA oligonucleotides were purchased from Dharmacon (Lafayette, CO).

DNA oligonucleotides Library construction for in vitro selection Pool-R: 5´-AACCTAAGGTCTCGTCGGCGCTATGGaaggaacagtgaattgacttcgtacgcgGACTGC GGTACCCCAGTAGGTACAGACAACGG-3´, where lower case letters indicate degenerate bases comprised of 91% of the original bases and 9% of three other bases (Pool-R1), or 79% of the original bases and 21% of three other bases (Pool-R2).

Pool-L: 5´-AGGGAAGGAAACTTCCCTGTGGAATTTTTGTAGTCNNNNNCCTAAGGCG CTTCGGCGAGACCAATCCAA-3´ (N indicates a mixed base); Fw-R: 5´-AACCTAAGGTCTCGTCGGCGCTA-3´ Rv-R: 5´-CCGTTGTCTGTACCTACTGGGGTACCGCA-3´

Fw-L: 5´-GAAGTAATACGACTCACTATTAGGGAAGGAAACTTCCCTGTGGAATTTTT-3´, where the T7φ2.5 promoter sequence is shown in italics; Rv-L: 5´-CCGTTGGTCCACATACGTATTTGGATTGGTCTCGCCGAAGCGCCTTAGG-3 ´

For in vitro selection cycles Rv-1: 5´-CCGTTGTCTGTACCTACTGGGGTACCGCA-3´ Rv-2: 5´-TCCACATACGTACCTGTCACGGTACCGCA-3´

1st Fw-1: 5´-CGTACACGTACTCACGCGTATACAGTC-3´ 1st Fw-2: 5´-ACTTCCGAGCTGTAGAGTTAGCAGCGA-3´ 2nd Fw-1: 5´-GAAGTAATACGACTCACTATTAGGGAAGGAAACTTCCCTGTGTCATTTTT-3´ 2nd Fw-2: 5´-GAAGTAATACGACTCACTATTAGGGAAGGAAACTTCCCTGTGGAATTTTT-3´ where the T7φ2.5 promoter sequence (11) is shown in italics;

3SSR-1:5´-GTACCCCAGTAGGTACAGACAACGG-3´/5´-CCGTTGTCTGTACCTA CTGGG-3´ 3SSR-2: 5´-GTACCGTGACAGGTACGTATGTGGA-3´/5´-TCCACATACGTACCTGTCACG -3´

5´-Biotin-modified RNA oligonucleotides Sub-1: 5´ biotin-CGUACACGUACTCACGCGUAUACAGUCCAC-3´;

Sub-2: 5´ biotin-ACUUCCGAGCUGUAGAGUUAGCAGCGACAC-3´

FAM-labeled oligonucleotide for activity assays 5´-FAM-CGTACACGTACTCACGCGTATACA-rGrUrCrCrArC-3´, where A, C, G, and T indicate deoxynucleotides and rA, rC, rG, and rU indicate ribonucleotides.

Stem-1

U U G U A A U U G

5'

A

G

G C G U C A G U A

U

U C C A G U C UC A

Stem-2

U

U

G

C

A

C C

C

G

A C G A

U U

G A

3'

C

UC

dG = -11.40 G

G

U

C A U G C

A

C

G C G U U A A

C G G A U U

U G

A C

U U

G A U A G A C U G C G

U U A

U

A

U 5'

G

A

3'

C C G

C U C U G A C C U

C

dG = -8.80

Figure S1. Possible secondary structures of 52nt-3’ ssr by mfold. (top) A stable structure with single-stranded 15nt-tail (red). The corresponding structure lacking stem-2 was also predicted for the 3’ ssr of the ∆16 mutant lacking the last 16 nucleotides (blue). (bottom) A less stable structure, in which 15nt-tail (red) forms base pairs with the last 16 nucleotides (blue). This type of structures is not predicted for the 3’ ssr of the ∆16 mutant lacking the last 16 nucleotides (blue).