Barcoded nucleotides

First publ. in: Angewandte Chemie International Edition ; 51 (2012), 1. - pp. 254-257
http://dx.doi.org/10.1002/anie.201105717
Barcoded NucIeotides**
Anna Baccaro, Anna-Lena Steck, and Andreas Marx*
DNA as an information storage syste m is simple and at the
same time complex owing to the va rious different arrangeme nts of the four natural nucleotides.1I1 The DNA synthesis
by DNA polyme rases is intriguing, since these enzymes are
able to catalyze the elongation of the primer strand by
recogni zing the DNA template and selecting the corresponding nucleotide.l lb.21 This feature can be exploited to diversify
th e four-base-code by substitution of the natural substrates
with modifi ed analogues PI Nucleotide analogues equipped
with various marker groups (e.g. dyes, tags, or spin labelsl41)
can be employed in DNA polymerase catalyzed reactions to
increase the application scope of DNA (e.g. sequ e ncing,
structural characterization, and immobilizationI4d.51). The
" information" embedded in the marker groups allow conclusions to be drawn from the evaluation of the resu lting
signals. A significant gain in information would result by
e mbedding a marker th at exhibits th e properties of a barcode.
Typically, th e ba rcode label bears no descriptive data but it
consists of a series of signs which code for th e de posited
information (the term was used in other contexts with DNA
before).16 1 For univ ersal adoption th e barcode should be
simple, affixed easily, and allow a relia bl e assignment of th e
deposited information . O ligodeoxynucleotides (ODNs) meet
the requirements of a barcode label to a great extent, since
the y have a simple code and can be distinguished by
characteristics such as self-assembly and hybridization specificity. For a simple introduction of th ese DNA barcode
labels into DNA, an e nzyme- mediated approach utilizing
ODN-modified nucleotides would be desirable. PI Howeve r,
the acceptance of th ese modified nucleotides by DNA
polyme rases should be hampe red by the steric demand of
th e ODN-modified nucleotides. Herein , we show that despite
the steric de mand th e e nzymatic synthesis of barcoded DNA
is feasible by usin g ODN-modified nucleoside triphosphates
that are a bout 40-times larger th a n the natural nucleo tides
and longer than the di a meter of a DNA polymerase (Figure 1 A).
["~I Dr. A. Baccaro,'+' Dipl.-Chem . A.-l. Steck,'+' Prof. Dr. A. Marx
Department of Chemi stry a nd Konstanz Resea rch School Chemi ca l
Biology, University of Konstanz
Uni versitatsstrasse 10, 78457 Konstanz (Germany)
E-m ail: andreas.marx@uni-konstanz.de
Homepage: http ://www.chemie.uni-kon stanz.de/ - agmarx/
[+1 Th ese authors contributed equa lly to this work .
[,'0'] We gratefully acknowledge support by the Konstanz Resea rch
School Chemica l Biology; the group of C. Hauck, Unive rsity of
Konstanz, for providin g equipment, and th e Mini sterium fur
Wissensc haft, Forschung und Kun st, Baden-Wurttemberg for
funding within the programm e Bionik.
A) dTIP
~
KienTaq
DNA polymera se
1 nm
B)H"~HL a} ~W.-"I-t.-v~
N-o
6~
•
'w',o, ..0_ 1 b} deprotection
~
I
A A
linker
""V"~o..~-aJ.n~
6
..,
barcode DNA strand
dT"'TP
H.l.o
"o,'P~
dT'TP
C) 6:
d(TTTTTT}
15/15a: d(AGG AAA GAA GAA TGG)
d(GTG GTI CAT ACT GGA}
15b:
20:
d(TTT TIA GGA AAG AAG AAT GG}
d(GAC CCA CTC CAT CGA GAT TIC TC}
23:
40:
d(TTT TTT TTT TGC TAA TIAAGC TIG GCT GCA GGT CGA CTI A}
Figure 1. A) Schematic depiction for th e comparison of size s. dTTP
versus dT,s'TP compared with KlenTaq DNA polym erase . B) Reaction
pathway for the synthesi s of ODN-modified dTTP. Yi elds and DNA
sequences are li sted in Table Sl of the Supporting Informat ion.
C) Sequences of barcode DNA strands . The numb ers indicate the
nucleotide lengths .
He rein, we introduce 2' -deoxyribonucleotide analogues,
containing an ODN at th e nucleobase (Figure 1 B), as
substrates for DNA polymerase mediated reactions. We
chose th e C5 position for pyrimidines and th e C7 position
for 7-deaza-purines to introduce the DNA strand at th e
nucleobase, since modifications at these positions have bee n
acce pted by DNA polymerases in several cases. I).HI To ODNbarcode-label nucleotides, an ODN strand was activated with
a commercial available carboxy modifie r at the 5' -end while
still on solid support and then coupled to the aminefunctiona lized triphosphates (Figure 1 B, see Supporting
Info rm ation) . After deprotection and cleavage from th e
solid support, th ese ODN-functionalized nucleotides were
tested in DNA polyme rase promoted primer-exte nsion reactions (yields and DNA sequ e nces are listed in Figure 1 C and
Supporting Information , Table Sl). We examined the acceptance of the ODN-modified thymidine a nalogues by DNA
polyme rases in primer-extension reactions (Figure 2A for
Therminalor DNA polymerase, Supporting Information Figure Sl for KlenTaq DNA polyme rase). We us ed a 23nucleotide (nt) primer with a 32P-label at th e 5'-end and a
35-nt templ ate, which contains a single A residue at position 27, coding for inserti on of a thymidine analogue aft er
ex te nding th e primer by three nucleo tides (Figure 2A).
Incub ati on with a DNA polymerase in absence of a thymidine
analogue res ulted in a prime r elonga tion that is predominantly paused at position 27 without generating significant
amounts of full-length product (Figure 2 A , lane 1), while the
reactio n including all four natural deoxynucleoside triphosphates (dNTPs) showed full -le ngth product (Figure 2A ,
254
Konstanzer Online-Publikations-System (KOPS)
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-173319
A)
5'-... TC
( dTll'
3'-... AG GGC ACG GTC GCG
B)
.
.
~
,. 1
~
primer
exlenslon I
J.~5
I annealing
--
.1- _
75nt
70nt
~'
- ---5'15
I
barcode primer """extension
42nt
35nt
~ ..
Figure 2. A) Partial DNA sequ ences of prim er and templ ate (see
Supportin g Inform ati on for more inform ati on) and PAGE analysis of
the prim er-exte nsion studi es usin g Th erminator DNA polym erase, a 23 nt prim er, a 3S-nt templ ate, and 10 flM dNTPs. M: DNA marker;
la ne 0 : 5'- 32 P-la beled prim er only; lane 1: prim er extension perform ed
in the presence of dATP, dCTP, a nd dGTP; lane 2: sam e as lane 1, but
in th e presence of dTIP ; lane 3: as lane 1, but in the prese nce of
dT' TP; lane 4: as lane 1, but in th e prese nce of dT 15' TP; lane 5: as
lane 1, but in the presence of dT" TP; lane 6: as lane 1, but in the
prese nce of dT' t>rP. 8) Elongation of one incorporated dT" MP. Left
sid e: Reacti on sequ ence used in thi s experiment (see Supportin g
Inform ation) . Ri ght sid e: PAG E analys is of the primer·extension
studi es usi ng KfenTaq DNA polymerase. M: DNA marker; lane 0 : 5'32 P·la beled prim er only, lane 1: primer-extension reaction I perform ed
in the presence of dATP, dCTP, dGTP, and dTIP; la ne 2: in th e
prese nce of dATP, dCTP, dGTP, and dT" TP; lane 3: barcod e prim erexte nsion reaction perform ed with natural dNTPs and unmodifi ed
DNA, lane 4: barcode primer-extension reacti on perform ed wi th natu ral dNTPs and dT" MP modified DNA.
lane 2) . By substitution of natura l thymidine with one of th e
modified triphosphates (dTTP, dT I5aTP, dT23TP or dT4'TP;
note: th e superscript numbers represent th e ODN-labe l
le ngth ; DNA sequenccs are listed in Figure 1 c and Supporting Information, Ta ble Sl) full -le ngth product was obtained
(Figure 2 A , lanes 3-6). Double bands were observ ed arising
from non -te mpla ted nucleotide ad dition to the 3'-te rmini of
th e blunt-ended DNA strand, which has been re po rted
before.191 As expected, these reacti on products mi grated
signifi ca ntly more slowly in de naturing polyacryl amide gel
electrophoresis (PAG E ) than the unmodifi ed full-l e ngth
reacti o n product, indicating that th e provided bulky nu cleotide is incorporated. The lowe r mobility th at increased with
the size of the label, is explained by th e additio nal bulk of the
incorpo ra ted barcode DNA stra nd . Simila r findin gs of lowe r
mobility for modified reaction products have been reported
before.IIO]
To e va luate the e ffici ency of incorporation of th e modi fi ed nucleo tid es in co mparison to the na tural nucleotides we
conducted single- nu cleotide incorpo ra ti o n expe rim e nts in
whi ch the modified nucleo tides (dT(T P, dT2'TP) directly
compete for incorpo ration with th e ir na tural counte rpa rts
(Supporting Informati on, Figure S2) . The ratio of unm odifi ed
versus modified nucleotide incorpo ration is easily accessible
by PAGE through th e signifi ca ntl y di ffe re nt rete nti o n times
ca used by th e incorpora ti on of th e bul ky m odifica tion . This
setup was pre viously used for the same purposel 8a] as we ll as to
st udy DNA polymerase selectivity.P I[ We found th a t Therminator DNA polyme rase inco rporates the investigated nucleotides with approximately 6- and 16-fold lower effi c iency than
th e natural nucleotide while for KlenTaq DNA p olymerase
33- and 66-fold lower effi ciencies were observed . The
obse rved effici e ncies compare we ll to rece ntly st udi e d C5modified dTIP analogues. lsal
We investigated th e feasibility of multiple inco rpo rations
(Supporting Informati on, Figure S3). Using dT2!YyP and a
template coding for the insertion of 46 TMPs in t he primer
exte nsion reaction , a highly branched reaction product is
generated with at least 7 modified nucleotides in a row.
E ncouraged by these res ul ts we synthesized dATP, dCTP, a nd
dGTP an alogues (see Supporting Informati o n) a nd tested
the m as well in the prime r extension reactio n (Figure S4). All
the analogues we re accepted by Therm inalor DNA po lyme rase and the prime r was extended to full -le ngth .
We tested th e ability of DNA poly me ra ses to utilize the
inco rp orated barcode DNA strand as a prime r in primer
exte nsion reactions. For this purpose, we pe rform ed prime r
exte nsion reactions with natural dNTPs as a control re action,
and ano th er reaction with dT23TP instead of dTIP using a 24nt primer a nd a 42-nt templa te coding for the inserti on of o ne
dTMP. These reaction products were hybridized with a seco nd
templ a te (69-nt) comple me ntary to the incorporate d ba rcode
DNA strand and incuba ted with a DNA polyme rase and
dNTPs for 1 h at 60 °C performing the barcode primer
exte nsio n reaction (Figure 2 B) . We obse rv ed comple te di sappea rance of th e initial band (Figure 2 B, lanes 2 a nd 4) and
the appea rance of a ne w band shifted to lower mobility,
indicating that the incorporated barcode DNA s trand was
used as the primer and elongated to a full-l e ngth prodpct
(Figure 2 B, la ne 4) . A s expected, in th e control reaction with
natural dNTPs, th e mobility of the reacti o n product aft er the
first prime r exte nsion was not altered on incubation und er the
same conditions. In addition, we tested the e lo ngation of the
inco rporated DNA stra nd by rolling circle amplification l121
(RCA) in solution a nd found extension as well (Supporting
Information, Figure S5).
. We investi ga ted wh eth er th e ODN-modifi ed nucl eotides
can be used as di agnostic tools for e nzy mati c reactions o n
solid suppo rts. The refore, we evalua ted the feasibility for th e
de tection of single nucl eotide variations in the seque nce
context of the B type Raf kin ase (BRAF) ge ne. The BRAF
soma tic T1 796A muta tion is e ncoun tered to a high ex te nt in
ma lignant me lano mas a nd human cancers.1131 Ge nome dissimilariti es, such as single nucleotide polymorphi sms (SNPs),
are ofte n responsibl e fo r a predisposition to th e diseasesP3. 141
and different drug effi cie nci es in ce rtain individua lsJlsl For th e
SNP detection system, primer probes were covalently bound
to a n amin o propyl PDITC (l,4-phe nyle ne diisothi ocya nate)
activ ated glass substra te. 1161 First single incorporati on of
ODN-modifi ed nucleo tides was performed using a templ ate
codin g for the insertion of a dTMP (Figure 3 A) . The refore,
two reaction blocks of nin e primer loci we re incubated in the
prese nce of a DNA polymerase, te mpl ate, and with dA ISTP or
dTISaT P. A fte r incubati o n, the slides were washed and
subseque ntl y incub ated wi th Cy3-labeled oligonucleotides
255
a nd seq uence-specific in trod ucti on of barcode O D N- labels by
e nzy mati c incorporatio n offers opportu niti es fo r future
applicati ons.
A)
B)
~~
ith
com: ,leme: e lc A
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Keywords: DNA pol ymerase· e nzym a ti c s yn t h esis · m icroarray·
nucl eot id es ' oli go n ucl eotid es
~Hyb"",,,,;on
glass
without complem
~
entary RCA primer
1'..
3'
S'
dNTP
DNAl!9.lymerase
s'
~
3'
Ol8sssllc\e
Figure 3, Microa rray-based sin gle- nucl eotid e-va riat ion detectio n
system. A) Reacti on sequ ence performed on DNA microa rray. Ri ght
side: Rea dou t at S32 nm aft er hybridi zation with Cy3-labeled oligonucleoti de. Reactions were co ndu cted under the sa me co ndi tions an d on
the sa me slide. B) Signa l amplifi cation by rollin g circl e amplifi cation .
Top: em ploying a complementary ci rcul ar DN A template. Botto m :
emp loying a non-co mp lementary circular DNA te mplate. Ri ght side:
Readout at 53 2 nm after hybridi zation with Cy3-labeled oligo nucleotides. Reacti ons we re co ndu cted und er the sa me co nditions and on
th e sa me slide.
tha t bind to the oligonucleotid e barcode of a n incorporated
dT ,s" MP. Clearly, an inte nse flu o resce nce signal was only
detected in cases where the ca nonical dT ,s"MP was incorporated. To investiga te signa l a mplificat ion we incub ated
ba rcode-modifi ed DNA complexes with a DN A polyme rase
in the prese nce of a ci rcul ar templa te th at binds to its
comple me nt ary ba rcode DNA stra nd (Figure 3 B) . The circula r template will e nable the ex tension of the co mp le mentary primer stra nd by mu ltiple copies of the seque nce e ncoded
in the templ ate by RCA. Subsequent ly, fo r signa l ge nerati o n
the slide was incubated wi th Cy3- modi fied o ligonucleo ti des.
As expected we could o bserv e significa nt signal increase only
at positio ns where barcodes compl ementa ry to the circul a r
templ a te we re prese nt.
Taken together, we in trod uce barcode-labele d dNTPs as
substrates fo r DNA polymerases. We showed that comme rcia ll y ava ilab le DNA polymerases a re ab le to process
modifie d nucleotides that are up to 40-times larger th a n the
natural substrate. The seq ue nce-specific incorporation of
barcode-modifi ed nucleotides a nd the ad dressa bi lity of
DNA by the sim ple hybridiza ti on of ca noni cal DNA stra nds
has potenti al fo r nume ro us applicati ons. T his met ho d is ve ry
adaptable, so d iffere nt techniques fo r fu rther DN A ma ni pulat io n a nd reado ut ca n be exploited, such as bio tin- st rep tavidin chemistry,1I6. 1na nopa rti cles,11 71or bra nched D NA amplifi ers (e_g. TSA detecti o n kit, bDN A amplifie r rtBI). T he system
has the pote nt ial to be expanded to a fo ur-color detecti on
system, using nucleoti de analogues ca rry ing uniqu e seque nces
a nd th e app ropri ate dye-labe led complementary DNA
strands. T he be nefi cia l combina ti o n of mi croa rray techniques
c.
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