Supporting Information for Walsh, Sachdeva & Silverman, J. Am. Chem. Soc. page S1

DNA Catalysts with Tyrosine Kinase Activity

Shannon M. Walsh, Amit Sachdeva, and Scott K. Silverman*

Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, United States

Table of Contents

Oligonucleotides, peptides, and DNA-anchored peptide conjugates ............................................. page S2 In vitro selection procedure ............................................................................................................ page S3 Selection progressions .................................................................................................................... page S6 Mass spectrometry and 8VP1 reactivity to establish phosphorylation product identities .............. page S7 Sequences of individual deoxyribozymes ...................................................................................... page S8 Single-turnover deoxyribozyme assay procedure .......................................................................... page S8 Additional data for deoxyribozyme kinetics and metal dependence .............................................. page S9 Dependence of deoxyribozyme activities on peptide and tether .................................................... page S13 Apparent Km(GTP) of 8EA101 and 16EC103 deoxyribozymes .................................................... page S15 References for Supporting Information .......................................................................................... page S16

OligonuclDNA

by solid-pylated RNtemplates with runnpreviouslyconjugatesterminal cas adapted

Figure S1.hexa(ethyle

Proce

5′-GGATAAdisulfide lDTT treatpH 7.5, anexcess DTproduct (Dacetate, pHdisulfide iby additioConjugatiand 5 µL the DNA-

Support

leotides, peptioligonucleoti

phase synthesNA (pppRNA

and T7 RNAning buffer 1×y.2 Peptides s were synth

cysteine side d from our rec

Synthesis andene glycol), or

edure for syntATACGACTCAlinker was intment. A 50 µnd 50 mM DTTT by additioDNA-HEG-p-H 7.0. Activain DMF and

on of 50 µL ofon to the pepof 20 mM peanchored pep

ting Informati

ides, and DNAides were obtsis on an ABIA) oligonucleA polymerase× TBE (89 m

were synthehesized by dischain of the pcent report.3

d structure of thHEG, tether c

thesis of DNAACTAT-HEG-pntroduced viaµL sample coTT was incubon of 50 µL o-C3-SH) was

ation as the pyincubating atf water, 10 µL

ptide was perfeptide (100 nmptide was puri

ion for Walsh

A-anchored ptained from InI 394 instrumotides were pe.1 All oligon

mM each Trisesized on thsulfide formapeptide, follo

he DNA-anchoonnecting the D

A-anchored pp-C3-SS-C3-Oa standard solontaining 5 nmbated at 37 °Cof water, 10 µdissolved in

yridyl disulfidt 37 °C for 2 L of 3 M NaCformed by admol, 20 equivified by 20%

h, Sachdeva &

peptide conjugntegrated DN

ment using reaprepared by

nucleotides w and boric ac

he solid phaation betweenowing the pro

ored CAAYAADNA oligonuc

peptide conjuOH-3′ (see filid-phase DNmol of DNA

C for 2 h. TheµL of 3 M Na35 µL of wat

de was achievh. The produ

Cl and 300 µLding 20 µL o

valents). The PAGE. A typ

Silverman, J.

gates NA Technologagents from Gin vitro tran

were purified bcid and 2 mMase as descrn a DNA HE

ocedure shown

A hexapeptide pcleotide anchor

ugate. The DNirst structure

NA synthesis anchor oligo

e reduced prodaCl, and 300 ter and 10 µL

ved by addinguct (DNA-HEL of ethanol aof 50 mM triesample was i

pical yield wa

J. Am. Chem. S

gies (CoralvilGlen Researc

nscription usiby 7 M urea

M EDTA, pHribed.3 DNAEG-tethered 3n in Figure S

phosphorylatior to the peptide

NA anchor oin Figure Sand unmask

onucleotide induct was precµL of ethano

L of 100 mM g 5 µL of 100 EG-p-C3-SSPyand dissolvedethylammoniuincubated at 3as 3.5–4.5 nm

Soc. p

lle, IA) or prech. 5′-Triphosng synthetic

a denaturing PH 8.3) as desc

A-anchored p3′-thiol and tS1 and given

on substrate. Ne.

ligonucleotidS1), where thked to a 3′-thn 100 mM HEcipitated to reol. The preciptriethylammomM 2,2′-dip

y) was precipd in 25 µL of wum acetate, pH37 °C for 12 h

mol.

age S2

epared sphor-DNA

PAGE cribed eptide the N-below

ote the

de was he 3′-

hiol by EPES, emove pitated onium

pyridyl pitated water. H 7.0, h, and

In vitro seThe se

as describ8VP1 deoFigure 2, a

Figure S2.of the 5′-trunwanted dat the α-phselections entirely; thnot base-paby Taq polprevious runphosphoGAATGAGGPAGE posversion (17rounds inclthe 5′-phosreaction paattachmentsequence tcapture olisuppressed

Support

election proceelection proc

bed previouslyoxyribozyme.and a depictio

. Nucleotide deriphosphorylatdeoxyribozymehosphorus atomfor which free

herefore, the leaired to anythilymerase and lreport for valiorylated Tyr (TGAAAGCACGAition, the lengt7 nt pppRNA, luding round 1sphorylated DNartners had dift of TyrOH to Rhat allows subigo, the 8VP1

d nonspecific cl

ting Informati

edure edure, cloniny,2a,4 but with5 An overvieon with nucle

etails of the seted RNA oligoes that catalyzem migrate diffe GTP replaceeft-hand DNA ing. In the reveleads to a size idation that 8yrOH).5 The seqAG-3′. To avoidth of the captueven-numbere). The pppRNA

NA componentfferent sequencRNA during thbstantial Watso1 binding armleavage (transe

ion for Walsh

ng, and initialh a different lew of the keyeotide details i

election and caonucleotide (3e covalent attacferently on PAd pppRNA as binding arm (ierse PCR primdifference bet

VP1 is highlyquence of the 8d emergence ofure oligo reactied rounds) andA-DNA chimet using a compces to avoid emhe capture stepon-Crick base

m was extendeesterification) o

h, Sachdeva &

analysis of iligation step3

y selection anis shown in F

apture steps of 6 nt pppRNAchment of the

AGE than doesthe phosphory

i.e., the pool smer, X is the H

tween the two y selective fo8VP1 catalyticf noncatalytic Don partner was

d a long versiora was prepare

plementary DNmergence of dp (this would pairing to the

ed several nuof the RNA nu

Silverman, J.

individual clo,5 and with a nd capture st

Figure S2.

in vitro selectA; phosphoryl

Tyr hydroxyl gs the desired cryl donor, the segment to the

HEG spacer (Gl PCR product

or reaction ofc region was 5′DNA sequences alternated in

on (54 nt pppRed by splint ligNA splint and Tdeoxyribozyme

be facilitated e constant captucleotides pastucleotides near

J. Am. Chem. S

ones were pernew capture

teps of each

tion. (A) Selecdonor) was chgroup to the RNcapture producRNA oligonuc5′-side of the

len Spacer 18)strands. (B) C

f phosphorylat-GGACACGATes that migratesuccessive rou

RNA-DNA chiation of the ppT4 DNA ligasees that catalyze

if the DNA rture oligo sequt the RNA-DNthe junction.

Soc. p

rformed essene step based oround is sho

tion step. The hosen such th

RNA via attackct of panel B. cleotide was orandom region

) that stops extCapture step. Sted Tyr (TyrP

GAGTGACTAAe aberrantly at aunds between amera, odd-num

ppRNA compone. The short ane unwanted corandom regionuence). For thNA junction,

age S3

ntially on the

own in

length

hat any of Tyr In the

omitted n) was tension See our P) over AGTG-a fixed a short

mbered nent to

nd long ovalent n had a he long

which

Supporting Information for Walsh, Sachdeva & Silverman, J. Am. Chem. Soc. page S4

Procedure for ligation step in round 1. The splint sequence was 5′-ATAGTGAGTCGTATTATCCTCCATC-AGGATCAGCTTAATACGACTCACTAT-3′, where the underlined T is included to account for the untemplated A nucleotide that is added at the 3′-end of each PCR product by Taq polymerase. This T nucleotide was omitted from the splint used for ligation of the initially random N40 pool, which was prepared by solid-phase synthesis without the untemplated A. A 35 µL sample containing 1 nmol of DNA pool, 800 pmol of DNA splint, and 500 pmol of 5′-phosphorylated DNA-anchored hexapeptide substrate was annealed in 5 mM Tris, pH 7.5, 15 mM NaCl, and 0.1 mM EDTA by heating at 95 °C for 3 min and cooling on ice for 5 min. To this solution was added 4 µL of 10× T4 DNA ligase buffer (400 mM Tris, pH 7.8, 100 mM MgCl2, and 5 mM ATP) and 1 µL of 5 U/µL T4 DNA ligase (Fermentas). The sample was incubated at 37 °C for 12 h and separated by 8% PAGE.

Procedure for ligation step in subsequent rounds. The same splint was used as in the round 1 ligation

procedure. A 17 µL sample containing the PCR-amplified DNA pool (~5–10 pmol), 25 pmol of DNA splint, and 50 pmol of 5′-phosphorylated DNA-anchored hexapeptide substrate was annealed in 5 mM Tris, pH 7.5, 15 mM NaCl, and 0.1 mM EDTA by heating at 95 °C for 3 min and cooling on ice for 5 min. To this solution was added 2 µL of 10× T4 DNA ligase buffer and 1 µL of 1 U/µL T4 DNA ligase (Fermentas). The sample was incubated at 37 °C for 12 h and separated by 8% PAGE.

Procedure for selection step in round 1. Each selection experiment was initiated with 200 pmol of the

ligated pool. A 20 µL sample containing 200 pmol of ligated pool and 300 pmol of 36 nt pppRNA phosphoryl donor was annealed in 5 mM HEPES, pH 7.5, 15 mM NaCl, and 0.1 mM EDTA by heating at 95 °C for 3 min and cooling on ice for 5 min. The selection reaction was initiated by bringing the sample to 40 µL total volume containing 70 mM HEPES, pH 7.5, 1 mM ZnCl2, 20 mM MnCl2, 40 mM MgCl2, and 150 mM NaCl. The Mn2+ was added from a 10× stock solution containing 200 mM MnCl2. The Zn2+ was added from a 10× stock solution containing 10 mM ZnCl2, 20 mM HNO3, and 200 mM HEPES at pH 7.5; this stock solution was freshly prepared from a 100× stock of 100 mM ZnCl2 in 200 mM HNO3. The metal ion stocks were added last to the final sample. For selections with GTP as the phosphoryl donor, the pppRNA was omitted and 1 mM GTP was added from a 10 mM stock solution after annealing. The sample was incubated at 37 °C for 14 h and separated by 8% PAGE.

Procedure for selection step in subsequent rounds. A 10 µL sample containing the ligated pool and 50

pmol of 36 nt pppRNA phosphoryl donor was annealed in 5 mM HEPES, pH 7.5, 15 mM NaCl, and 0.1 mM EDTA by heating at 95 °C for 3 min and cooling on ice for 5 min. The selection reaction was initiated by bringing the sample to 20 µL total volume containing 70 mM HEPES, pH 7.5, 1 mM ZnCl2, 20 mM MnCl2, 40 mM MgCl2, and 150 mM NaCl. For selections with GTP as the phosphoryl donor, the pppRNA was omitted and 1 mM GTP was added from a 10 mM stock solution after annealing. The sample was incubated at 37 °C for 14 h and separated by 8% PAGE.

Procedure for capture step in round 1. A 28 µL sample containing ligated pool, 500 pmol of 54 nt 5′-

triphosphorylated RNA-DNA chimera substrate, and 300 pmol of 8VP1 capture deoxyribozyme was annealed in 5 mM HEPES, pH 7.5, 15 mM NaCl, and 0.1 mM EDTA by heating at 95 °C for 3 min and cooling on ice for 5 min. The capture reaction was initiated by bringing the sample to 40 µL total volume containing 50 mM HEPES, pH 7.5, 20 mM MnCl2, and 150 mM NaCl. The sample was incubated at 37 °C for 14 h. Before PAGE, to the sample was added 500 pmol of a 60 nt decoy oligonucleotide complementary to the 40 nt 8VP1 catalytic region and 10 nt of binding arm on each side, to ensure complete removal of the capture deoxyribozyme from the pool. The sample was separated by 8% PAGE.

Procedure for capture step in subsequent rounds. A 14 µL sample containing ligated pool, 50 pmol of

17 nt pppRNA substrate (in even rounds) or 54 nt 5′-triphosphorylated RNA-DNA chimera substrate (in odd rounds), and 25 pmol of 8VP1 capture deoxyribozyme was annealed in 5 mM HEPES, pH 7.5, 15 mM NaCl, and 0.1 mM EDTA by heating at 95 °C for 3 min and cooling on ice for 5 min. The capture

Supporting Information for Walsh, Sachdeva & Silverman, J. Am. Chem. Soc. page S5

reaction was initiated by bringing the sample to 20 µL total volume containing 50 mM HEPES, pH 7.5, 20 mM MnCl2, and 150 mM NaCl. The sample was incubated at 37 °C for 14 h. To the sample was added 100 pmol of a 60 nt decoy oligonucleotide complementary to the 40 nt 8VP1 catalytic region and 10 nt of binding arm on each side (note that the decoy sequence is different in even-numbered and odd-numbered rounds). The product was separated by 8% PAGE.

Procedure for PCR in subsequent rounds. In each selection round, two PCR reactions were

performed, 10-cycle PCR followed by 30-cycle PCR. First, a 100 µL sample was prepared containing the PAGE-separated capture product, 200 pmol of forward primer, 50 pmol of reverse primer, 20 nmol of each dNTP, and 10 µL of 10× Taq polymerase buffer (200 mM Tris-HCl, pH 8.8, 100 mM (NH4)2SO4, 100 mM KCl, 20 mM MgSO4, and 1% Triton X-100). This sample was cycled 10 times according to the following PCR program: 94 °C for 2 min, 10× (94 °C for 30 s, 47 °C for 30 s, 72 °C for 30 s), 72 °C for 5 min. Taq polymerase was removed by phenol/chloroform extraction. Second, a 50 µL sample was prepared containing 1 µL of the 10-cycle PCR product, 100 pmol of forward primer, 25 pmol of reverse primer, 10 nmol of each dNTP, 20 µCi of α-32P-dCTP (800 Ci/mmol), and 5 µL of 10× Taq polymerase buffer. This sample was cycled 30 times according to the following PCR program: 94 °C for 2 min, 30× (94 °C for 30 s, 47 °C for 30 s, 72 °C for 30 s), 72 °C for 5 min. Samples were separated by 8% PAGE.

Selection

Figure S38VP1-catalthe yield fcloned rounrandom regCAAYOHAphosphorylactivity wa

Support

progressions

. Progressions lyzed capture r

for the 8VP1-cnds. The CF1, gion, respectiv

AA phosphoryll donor. For th

as undetectable

ting Informati

of the in vitroreaction using tcatalyzed captuCH1, and CK

vely, and pppRlation using a

he EB1 and ECe (<0.5%; 16EB

ion for Walsh

o selection expthe CAAYPAAure reaction us1 selections we

RNA as the phan N30, N40, aC1 selections, tB1 and 8EC1).

h, Sachdeva &

periments. In A substrate as ising the deoxyere for CAAYO

hosphoryl donoand N50 randothe activities a

Silverman, J.

each round, “cillustrated in Fiyribozyme pooOHAA phosphoor. The EA1, Eom region, re

are shown begi

J. Am. Chem. S

control” refersigure S2B, and

ol for that rounorylation usingEB1, and EC1

espectively, aninning with the

Soc. p

s to the yield fd “selection” rend. Arrows mag an N30, N40, a1 selections wend free GTP e last round in

age S6

for the efers to ark the and N50 ere for as the which

Mass spec

Table S1. Masses areproduct wasubstrate (annealed inice for 5 mfrom a 10 mthe sample and 150 mfourth to oPAGE-puriUltrafleXtrPAGE and the plot, esmM HEPEthat the tyindicated k

Support

ctrometry and

MALDI masse tabulated foas prepared frFigure S1), 3n 5 mM HEPE

min. For samplemM stock soluto 20 µL total

mM NaCl. The ne-half of the ified sample wreme; matrix 3subjected to 8

ssentially the sES, pH 7.5, 20 yrosine hydroxykinase deoxyrib

ting Informati

d 8VP1 reactiv

deoxyribo

6CF16CF17CH17CH18CK18CK18EA1

21EB116EC1

s spectrometryor the DNA-Hom a 10 µL s50 pmol of d

ES, pH 7.5, 15 es with GTP asution after annel volume contasample was inproduct band

was desalted by3-hydroxypicol8VP1-catalyzedsame rate and mM MnCl2, ayl group is th

bozyme.

ion for Walsh

vity to establi

ozyme

27 34 02 04 05 28 01

121 103

y and 8VP1 reHEG-CAAYPAsample contain

deoxyribozymemM NaCl, ands the phosphorealing. The DNaining 70 mM Hncubated at 37 was excised, ty Millipore C1

linic acid). Foud reaction withyield of produ

and 150 mM Nhe attachment

h, Sachdeva &

ish phosphory

[M+H]+ calcd.

[

6982.9 66982.9 66982.9 66982.9 66982.9 66982.9 66982.9 66982.9 66982.9 6

eactivity data AA product. Tning 300 pmoe, and 400 pmd 0.1 mM EDTryl donor, the p

NA-catalyzed pHEPES, pH 7.5°C for 14 h a

to suppress ext8 ZipTip and a

ur of the indivh the pppRNA cuct formation w

NaCl at 37 °C; site for the ph

Silverman, J.

ylation produ

[M+H]+

found e

(fou

6982.6 –6982.0 6981.7 6984.8 6982.7 –6981.4 6984.7 6982.2 6986.3

for individualThe DNA-anch

l of DNA-ancmol of pppRNTA by heatingpppRNA was o

phosphorylation5, 0.5 mM ZnC

and separated btraneous signaanalyzed by M

vidual phosphocapture oligonwere observedkobs ca. 0.2 h–

hosphoryl grou

J. Am. Chem. S

uct identities

error, % nd – calcd.) –0.004 –0.01 –0.02 +0.03 –0.003 –0.02 +0.03 –0.01 +0.05

l tyrosine kinahored CAAYPAchored HEG-te

NA phosphorylg at 95 °C for 3omitted and 1 n reaction was Cl2, 20 mM Mby 8% PAGE als due to unreMALDI mass sorylation produnucleotide. In ed as for the YP

1). These data up that was jo

Soc. p

ase deoxyribozAA phosphoryethered CAAYl donor, which3 min and coolmM GTP wasinitiated by brnCl2, 40 mM M(only the loweacted substratespectrometry (Bucts were isolaeach case as shoP positive contrdirectly demo

oined to YOH

age S7

zymes. ylation

YOHAA h were ling on added ringing MgCl2, er one-e). The Bruker ated by own in rol (50 nstrate by the

Sequences

Figure S4deoxyribozspecific x nAll alignmuppermost the far righalignment Alignment nt segmentthat use GT Single-tur

The polynucleMgCl2, ananchored donor wasmin and cand 2 µL DNA-catacontainingother ion mM MnC100 mM Hin 200 mMaliquots thµL stop so50 mM EPAGE anddata direcErrors in k

Support

s of individua

4. Sequences zymes were usnucleotides (x ents show onlysequence; a d

ht is shown (inof all initiallywas performe

t present at theTP as the phosp

rnover deoxyrDNA-anchorotide kinase nd 1 mM spphosphopept

s annealed in ooling on iceof 1 mM GT

alyzed phosphg 70 mM HEPconcentration

Cl2. The Zn2+ HEPES at pHM HNO3. Thehat were all inolution (80%

EDTA, 0.025d quantified wtly to first-orkobs values w

ting Informati

al deoxyriboz

of the initiased as 5′-CGA= 30, 40, or 50y the initially rash denotes a n parentheses) y obtained N30

ed using CLC Se 5′-end of the phoryl donor.

ribozyme assared hexapept(Fermentas),

permidine). Atide substrate5 mM HEPE

e for 5 min. FoTP was addedhorylation rePES, pH 7.5, ns as approprwas added fr

H 7.5; this stoe metal ion stncubated at 3formamide,

% bromophewith a Phosphrder kinetics; ere calculated

ion for Walsh

ymes

ally random AAGCGCTAGAA0) of the initialrandom regiongap. In both sethe number of, N40, and N50

Sequence Viewsequences (bo

ay proceduretide substrat using 10× b

A 10 µL same, 10 pmol ofES, pH 7.5, 15or assays withd from a 10

eaction was i0.5 mM ZnC

riate). The Mrom a 10× stock solution wtocks were ad37 °C. At app1× TBE [89

enol blue, 0.0horImager. Vai.e., yield Y =

d as the stand

h, Sachdeva &

regions of thACAT-Nx-ATAlly random reg. A dot denoteets of sequencf times that se0 deoxyribozymwer using stanoxed in grey).

te was 5′-32

buffer that lample containinf deoxyriboz5 mM NaCl, ah GTP as the mM stock sonitiated by b

Cl2, 20 mM MMn2+ was adde

ock solution cwas freshly prdded last to thpropriate timemM each Tri025% xylenealues of kobs w= Ymax•(1 – e–

dard deviation

Silverman, J.

he new tyrosAGTGAGTCGTAgion as determes conservationes, next to the

equence was fomes that use pdard paramete(B) Unaligned

2P-radiolabeleacks DTT (50ng 0.25 pmo

zyme, and 20and 0.1 mM Ephosphoryl d

olution after abringing the

MnCl2, 40 mMed from a 10×containing 5repared from he final sample points, 2 µLis and boric ae cyanol). Sawere obtained–kt), where k =n from the in

J. Am. Chem. S

sine kinase dATTA-3′, wher

mined through tn, i.e., the samee sequence lengound during clpppRNA as thers. Note the std sequences of

ed using γ-00 mM Tris,ol of 5′-32P-r0 pmol of ppEDTA by headonor, the pppannealing in sample to 20

M MgCl2, and× stock solutmM ZnCl2, 1a 200× stockle, which was

L aliquots weracid and 2 mMamples were d by fitting th= kobs and Yma

ndicated numb

Soc. p

deoxyribozymere Nx representhe selection pre nucleotide asgth in nucleotiloning. (A) Seqhe phosphoryl trongly conservf the deoxyribo

-32P-ATP an, pH 7.6, 100radiolabeled DppRNA phospating at 95 °CpRNA was om8 µL volume

0 µL total vod 150 mM Naion containin10 mM HNOk of 100 mM s divided intore quenched wM EDTA, pHseparated by

he yield versuax is the final

mber of indepe

age S8

es. All nts the rocess. s in the ides on quence donor. ved 11

ozymes

nd T4 0 mM DNA-phoryl

C for 3 mitted e. The olume

aCl (or ng 200 O3, and

ZnCl2 o 2 µL with 5

H 8.3], y 20% us time

yield. endent

determinapoints wer Additiona

Figure S5.conditions:for the six were as fol1.42. 7CH8CK101 00.10, and 8

Support

ations. When re fit to a stra

al data for deo

. Kinetic data : 70 mM HEPEdeoxyribozymllows. 6CF1 de1 deoxyribozy.20, 8CK105 0

8CK133 0.15.

ting Informati

kobs was suffiaight line, and

oxyribozyme k

for tyrosine kiES, pH 7.5, 0.5

mes of Figure 3eoxyribozymes

ymes (N40): 7C0.27, 8CK106

ion for Walsh

iciently low sd kobs was take

kinetics and m

inase deoxyrib5 mM ZnCl2, 20 are included fs (N30): 6CF10

CH102 0.12, 7C0.15, 8CK109

h, Sachdeva &

uch that an een as the slope

metal depend

ozymes that u0 mM MnCl2, for completene01 0.26, 6CF10CH104 0.16, a9 0.11, 8CK11

Silverman, J.

exponential fite of the line.

dence

use pppRNA as40 mM MgCl2

ess and to facil03 0.21, 6CF12and 7CH106 016 0.30, 8CK1

J. Am. Chem. S

t was not mea

s the phosphor2, and 150 mMlitate comparis25 019, 6CF12

0.14. 8CK1 de121 0.085, 8C

Soc. p

aningful, the

ryl donor. IncuM NaCl at 37 °C

sons. kobs value27 0.52, and 6eoxyribozymes

CK128 0.15, 8C

age S9

initial

ubation C. Data es (h–1) CF134 (N50): CK129

Figure S6.Incubation as indicatecombinatiodeoxyriboz

Support

. Metal dependconditions: 70

ed, and 150 mon Mn/Mg hazymes of Figur

ting Informati

dence data for 0 mM HEPES,mM NaCl at 3ad no activityre 3B.

ion for Walsh

tyrosine kinas, pH 7.5, comb37 °C. (A) Day in all case

h, Sachdeva &

se deoxyribozybinations of 0.5ata at t = 24 hs (<0.5%; da

Silverman, J.

ymes that use 5 mM ZnCl2, 2

h as in Figure ata not shown

J. Am. Chem. S

pppRNA as th20 mM MnCl2

3B for all dn). (B) Kinetic

Soc. pa

he phosphoryl 2, and 40 mM

deoxyribozymec plots for th

ge S10

donor. MgCl2

es. The he six

Figure S7HEPES, pHbuffer contwith GTP);different dGTP as the

Support

. Dependence H 7.5, 0–3 mMtributed a fina; the Zn2+ concata sets. (A) D

e phosphoryl do

ting Informati

of tyrosine kinM ZnCl2, 20 mM

l EDTA concecentration in thDeoxyribozymeonor.

ion for Walsh

nase deoxyriboM MnCl2, 40 mentration of 0.0he plots was unes that use ppp

h, Sachdeva &

ozymes on ZnmM MgCl2, and05 mM (for sa

ncorrected for EpRNA as the p

Silverman, J.

n2+ concentratid 150 mM NaCamples with ppEDTA chelatiophosphoryl do

J. Am. Chem. S

on. IncubationCl at 37 °C forppRNA) or 0.

on. Different syonor. (B) Deox

Soc. pa

n conditions: 7r 24 h. The ann04 mM (for saymbols correspxyribozymes th

ge S11

70 mM nealing amples pond to hat use

Figure S8HEPES at NaCl at 37of kobs on bis challengslope is bet

Support

. pH dependenthe indicated p

7 °C. Each databuffer compouning. The straigtween 1.0 and

ting Informati

nce data for thpH for the 1 Ma point is plottend identity (comght lines are fo1.5.

ion for Walsh

he 6CF134 deoM stock solution

ed as mean ± smpare MES ve

or reference and

h, Sachdeva &

oxyribozyme. n, 0.5 mM ZnCstandard deviatersus MOPS atd have slopes

Silverman, J.

Incubation conCl2, 20 mM Mtion (n = 4). Bt pH 6.5), assigof 0.5, 1.0, 1.5

J. Am. Chem. S

nditions: 70 m

MnCl2, 40 mM Because of the gning a single s5, and 2.0. The

Soc. pa

mM MOPS, MMgCl2, and 15apparent depenslope from these best estimate

ge S12

MES, or 50 mM ndence se data

e of the

Dependen

Figure S9.HEPES, pHdependencedeoxyribozsimilarly toboth positiare found strand;6 herlength depeDNA 3′-hydeoxyribozselective amodestly sselective am6CF134: 1.

Support

nce of deoxyri

. Dependence H 7.5, 0.5 mMe. The substratzymes of Figuo 6CF134; all ons flanking that the N-termire this sequencendence. The ydroxyl group zymes, which mong the threelective for themong the three.23, 2.09, 0.80,

ting Informati

ibozyme activ

of deoxyribozyM ZnCl2, 20 mM

te included theure 3 (data for

three deoxyribhe Tyr (Y) resinus of the nace serves as a substrate inclufor phosphoryare representa

ee peptide lenge longer parene peptide lengt, 0.012. 7CH10

ion for Walsh

vities on pept

yme activity oM MnCl2, 40 me indicated pepr 6CF134 and bozymes tolerasidue. The subsatural human rh

test substrate uded the indicaylation. Full kiative of the thgths and also t CAAYAA suths). kobs value02: 0.13, 0.071

h, Sachdeva &

tide and tether

on peptide sequmM MgCl2, antide on a HEG7CH102 are a

ate each of Phestrate with GPhinovirus 14 sthat has a Tyr

ated peptide oninetic plots arehree types of slightly activeubstrate over thes (h–1) were a1, 0.047, ~0. 8C

Silverman, J.

r

uence and lengnd 150 mM NaG tether (t = 24 also in Figuree (F), Glu (E),

PYSGN was evsequence, wher flanked by mn a HEG tethee shown for thbehavior illu

e with the DNhe shorter CYA

as follows, listeCK105: 0.25, 0

J. Am. Chem. S

gth. IncubationaCl at 37 °C. ( h). Data are s

e 4). 6CF127 a, or Lys (K) invaluated becauere the Tyr is many non-Ala rer, or the substhe 6CF134, 7C

ustrated in FigNA 3′-hydroxyl

A and CY subed top to botto0.20, 0.17, ~0.

Soc. pa

n conditions: 7(A) Peptide seqhown here for and 8CK105 bn place of Ala use these amino

attached to anresidues. (B) Ptrate provided CH102, and 8Cgure 4 (6CF13l substrate; 7C

bstrates; 8CK10om for each da

ge S13

70 mM quence all six

behave (A) at

o acids n RNA Peptide only a

CK105 34: not CH102: 05: not ata set.

Figure S10hexapeptidand 150 m(C) 7CH10

Support

0. Dependencede substrate. IncmM NaCl at 302 and 7CH104

ting Informati

of deoxyribozcubation condi37 °C. (A) Str4 deoxyribozym

ion for Walsh

zyme activity oitions: 70 mM ructures of themes. (D) 8CK1

h, Sachdeva &

on length of tethHEPES, pH 7.

e three substra105 and 8CK12

Silverman, J.

ther connecting.5, 0.5 mM ZnCates. (B) 6CF128 deoxyribozy

J. Am. Chem. S

g the DNA ancCl2, 20 mM M127 and 6CF1ymes.

Soc. pa

chor to the CAA

MnCl2, 40 mM M34 deoxyriboz

ge S14

AYAA MgCl2, zymes.

Apparent

Figure S11was assaye150 mM Nplots of ymean ± stankobs values left-hand pfrom initiavalue (i.e., productive coefficientsn = 1 and mn and m. (However, owe conserv16EC103. other combconservativ

Support

Km(GTP) of 8

1. Determininged under incubNaCl, and 0.1–

ield versus timndard deviatiofrom initial-ra

plot in Figure 5al-rate kinetics.

kobs at saturatiGTP binding

s for productivm = 3; the corrA) 8EA101. Fother combinatvatively estimaFitting both abinations of apvely estimate th

ting Informati

8EA101 and

g the apparent Kation condition1000 µM GTPme from 0 to

on, noting that ate kinetics are 5), but all of th. Data were fiing GTP conceg, Ki is the ive and unprodresponding fit vFitting both apptions of appareate that appareapparent K valupparent Km anhat apparent Km

ion for Walsh

16EC103 deo

Km(GTP) of thens of 70 mM HP at 37 °C, witho 2 h; n = 3 some error baslightly differ

he apparent Km

t to kobs = kcat•entration), C isinhibition conductive GTP bvalues of appaparent K valueent Km and Ki aent Km is betwues simultaneo

nd Ki also ledKm is between 2

h, Sachdeva &

oxyribozymes

e 8EA101 and HEPES, pH 7.5h kobs values dat each GTP

ars are smaller ent from those

m values were d[Cn/(Km

n+Cn)]s the GTP concnstant for unp

inding, respecarent Km and Kes simultaneoualso led to acce

ween 12–32 µMously gave Km

to acceptable6–72 µM and a

Silverman, J.

s

16EC103 deox5, 0.5 mM ZnCdetermined from

concentrationthan the sizes

e obtained by fdetermined usi•[1–Cm/(Ki

m+Ccentration, Km

roductive GTctively. Data fo

Ki did not depenusly gave Km =eptable curve f

M and apparenm = 42 ± 7 µM e curve fits, aapparent Ki is b

J. Am. Chem. S

xyribozymes. E

Cl2, 20 mM Mnm initial-rate k

n). The kobs vas of the data pfitting the full ping internally c

Cm)], where kca

is the apparenP binding, an

for each deoxynd strongly on = 20 ± 1 µM afits, as illustratnt Ki is betwee and Ki = 284 s illustrated. Fbetween 245-3

Soc. pa

Each deoxyribnCl2, 40 mM Mkinetics (linearalues are plotoints. The numprogress curveconsistent kobs

at is the apparent Km(GTP) vand n and m aryribozyme fit w

the precise vaand Ki = 240 ±ted. From thesen 200–290 µM± 17 µM. Ho

From these da330 µM.

ge S15

bozyme MgCl2, r fits to tted as merical es (e.g.,

values ent kcat

alue for re Hill well to lues of 4 µM.

se data, M. (B) wever,

ata, we

Supporting Information for Walsh, Sachdeva & Silverman, J. Am. Chem. Soc. page S16

References for Supporting Information (1) Milligan, J. F.; Groebe, D. R.; Witherell, G. W.; Uhlenbeck, O. C. Nucleic Acids Res. 1987, 15, 8783-

8798. (2) (a) Flynn-Charlebois, A.; Wang, Y.; Prior, T. K.; Rashid, I.; Hoadley, K. A.; Coppins, R. L.; Wolf, A.

C.; Silverman, S. K. J. Am. Chem. Soc. 2003, 125, 2444-2454. (b) Wang, Y.; Silverman, S. K. Biochemistry 2003, 42, 15252-15263.

(3) Chandrasekar, J.; Silverman, S. K. Proc. Natl. Acad. Sci. USA 2013, 110, 5315-5320. (4) Kost, D. M.; Gerdt, J. P.; Pradeepkumar, P. I.; Silverman, S. K. Org. Biomol. Chem. 2008, 6, 4391-

4398. (5) Sachdeva, A.; Chandra, M.; Chandrasekar, J.; Silverman, S. K. ChemBioChem 2012, 13, 654-657. (6) (a) Stanway, G.; Hughes, P. J.; Mountford, R. C.; Minor, P. D.; Almond, J. W. Nucleic Acids Res.

1984, 12, 7859-7875. (b) Werner, G.; Rosenwirth, B.; Bauer, E.; Seifert, J. M.; Werner, F. J.; Besemer, J. J. Virol. 1986, 57, 1084-1093.