University of Groningen

Synthesis of DNA block copolymers with extended nucleic acid segments by enzymaticligationAyaz, Meryem S.; Kwak, Minseok; Alemdaroglu, Fikri E.; Wang, Jie; Berger, Ruediger;Herrmann, Andreas; Berger, RüdigerPublished in:Chemical Communications

DOI:10.1039/c0cc04746e

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Citation for published version (APA):Ayaz, M. S., Kwak, M., Alemdaroglu, F. E., Wang, J., Berger, R., Herrmann, A., & Berger, R. (2011).Synthesis of DNA block copolymers with extended nucleic acid segments by enzymatic ligation: cut andpaste large hybrid architectures. Chemical Communications, 47(8), 2243-2245.https://doi.org/10.1039/c0cc04746e

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ISSN 1359-7345

Chemical Communications

www.rsc.org/chemcomm Volume 47 | Number 8 | 28 February 2011 | Pages 2181–2456

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COMMUNICATIONAndreas Herrmann et al.Synthesis of DNA block copolymers with extended nucleic acid segments by enzymatic ligation: cut and paste large hybrid architectures 1359-7345(2011)47:8;1-8

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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 2243–2245 2243

Cite this: Chem. Commun., 2011, 47, 2243–2245

Synthesis of DNA block copolymers with extended nucleic acid segments

by enzymatic ligation: cut and paste large hybrid architecturesw

Meryem S. Ayaz,za Minseok Kwak,zb Fikri E. Alemdaroglu,yb Jie Wang,aRudiger Berger

a

and Andreas Herrmann*b

Received 2nd November 2010, Accepted 9th December 2010

DOI: 10.1039/c0cc04746e

Ultra-high molecular weight DNA/polymer hybrid materials

were prepared employing molecular biology techniques. Nucleic

acid restriction and ligation enzymes were used to generate

linear DNA di- and triblock copolymers that contain up to

thousands of base pairs in the DNA segments.

Starting in the late twentieth century, DNA has doubtlessly been

one of the most extensively studied biopolymers, not only for its

critical biological role but for its unique and intriguing structural

properties. For instance, it has famously been utilized for the self-

assembly of complex and beautiful nanostructures.1,2 The same

self-recognition properties have also been widely investigated in

nucleic acid hybrid materials as a powerful tool to alter material

structure and other properties on demand. In a medical context,

an RNA aptamer covalently bonded with poly(ethylene glycol)

(PEG), known as pegaptanipt, is already available in clinics for

the treatment of age-related macular degeneration of the retina.3

Indeed, the pairing of oligodeoxynucleotides (ODNs) with

synthetic polymers has far-reaching potential applications, from

drug delivery and diagnostics to nanotechnology.4

The initial synthetic approach for these tailor-made materials

was coupling a polymer to an ODN in solution. However,

covalent bond formation between hydrophobic polymers and

ODNs was generally limited due to their solvent incompatibility.

One innovative method to overcome this barrier is solid phase

coupling, in which the hydrophobic units are attached to

elongated ODNs immobilized on resin. This technique has

yielded amphiphilic DNA block copolymers (DBCs) bearing

poly(styrene) or poly(propylene oxide) (PPO) blocks.5,6 More

recently, a method from molecular biology has been applied to

the generation of DNA–polymer hybrids in order to attain longer

ODN blocks than those possible through chemical synthesis

(B200 bases). Namely, the polymerase chain reaction (PCR),

when performed using plasmid DNA and either di- or triblock

DBCs as the primer, enabled the production of well-defined

multiblock copolymers with double-stranded (ds) DNA blocks

of B1578 base pairs (bp) and a total number average molecular

weight (Mn) of more than 1000 kDa, including the polymer.7 The

introduction of a thermoresponsive polymer, poly(N-isopropyl-

acrylamide) (PNIPAM), into the primer in this technique allowed

the rapid and facile isolation of the specific, desired amplicons

from the complex reaction mixture.7

Here we present a novel synthetic strategy for the prepara-

tion of ultra-high molecular weight di- and triblock DBCs with

even longer DNA blocks using another molecular biology

technique, enzymatic restriction and ligation. Just as one

builds paper models by cutting and pasting paper strips, we

have prepared large polymer–DNA architectures by digestion

and ligation of DNA using the corresponding enzymes as tools

(Fig. 1). The family of hydrophilic and hydrophobic polymers

used in this study proves the feasibility of this method to

extend DBCs by different lengths of dsDNA, from 800 to

2300 bp, yielding highly defined block architectures with

molecular weights in the range of three million dalton.

The five single-stranded (ss) di- and triblock DBCs used as

starting materials in this study were prepared by solution

coupling or solid phase synthesis, as previously reported by

our group (Fig. 1A).7,8 The diblock architectures (ODN-b-

polymer) consisted of a 22mer ODN segment (see Table S1,

ESIw for the sequence) where the 50-end is covalently bonded

to a polymer block, whether PEG (Mn = 5 kDa or 20 kDa,

ssDB1 or ssDB2), PNIPAM (Mn = B6 kDa, ssDB3) or PPO

(Mn = 6.8 kDa, ssDB4). In the triblock architecture, a PEG

(Mn = 4 kDa) block was conjugated at both termini to

identical ssODNs (ODN-b-PEG-b-ODN, ssTB).

To prepare the longer DNA segments for the extension

of the ss copolymers above, a circular plasmid DNA (pBR322,

4361 bp) was digested by a DNA restriction enzyme (Alw26I)

into three dsDNA segments, named short (S, 772 bp), middle

(M, 1311 bp) and long (L, 2279 bp). The different fragments

were isolated by agarose gel electrophoresis (Fig. 1B). Each gel

band of the restriction products was subsequently purified

using a gel extraction kit.

Hybridization of DNA block copolymers (ssDB1-4) with

complementary sequences designed to introduce short over-

hangs and ligation with the restricted dsDNA segments were

aMax Planck Institute for Polymer Research, Ackermannweg 10,55128 Mainz, Germany

bDepartment of Polymer Chemistry, Zernike Institute for AdvancedMaterials, University of Groningen, Nijenborgh 4,9747 AG Groningen, The Netherlands. E-mail: a.herrmann@rug.nl;Fax: +31 50 363 4400; Tel: +31 50 363 6318

w Electronic supplementary information (ESI) available: Experimentaland analytical details. See DOI: 10.1039/c0cc04746ez These authors contributed equally.y Current address: BASF Poliuretan Sanayi, Seyhli Mh. Ankara,34912, Istanbul, Turkey.

ChemComm Dynamic Article Links

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2244 Chem. Commun., 2011, 47, 2243–2245 This journal is c The Royal Society of Chemistry 2011

carried out in one pot through simple mixing and incubation.

Each complementary DNA (cDNA-S, cDNA-M and cDNA-L,

respectively) can be hybridized with any of the DBCs used here

because the 22mer DNA blocks are the same in each. In

addition to a segment complementary to the DBC 22mer

sequence, each cDNA contains an additional four-nucleotide

overhang at the phosphorylated 50-end for recognition of the

sticky end of the desired dsDNA segment. These three DNA

components, i.e. ssDB, restriction products for extension

(S, M or L) and the corresponding cDNA, were mixed in the

presence of T4 DNA ligase, adenosine triphosphate (ATP) and

hybridization buffer and incubated at 16 1C for 48 h (see ESI.2wfor experimental details). This ligation resulted in the controlled

extension of the DNA blocks (S/M/L-dsDB1-4, Fig. 1C).

After gel extraction of the ligation products, 15 different

products and controls were characterized by agarose gel electro-

phoresis under ethidium bromide (EtBr) staining (Fig. 2). First, it

should be noted that all bands were discrete through the entire

gel, demonstrating that the materials thus produced have high

purity. The success of the ligation is clear from the trend in

electrophoretic mobility, which, as anticipated, was dominated

by the length of the extended DNA segments. A secondary

determining factor in the mobility was the nature of the polymer

attached. The widest variation due to the hydrophobicity and

molecular weight of the appended polymer was observed

for the materials extended with the shortest segments (group S

in Fig. 2A), all of which exhibited the fastest mobilities.

Interestingly, the bands of the three hydrophilic polymers

(S-dsDB1 to 3, lanes S1–S3 in Fig. 2A) didn’t differ significantly

although the Mn of the PEG segment in S-dsDB2 is four times

higher than that of S-dsDB1. Moreover, the PNIPAM conjugate

(S-dsDB3) had the lowest electrophoretic mobility of the hydro-

philic polymer architectures in this group in spite of the low Mn

of PNIPAM used (6 kDa). Indeed, the markedly lower mobility

of the PPO-containing material (lane S4, S-dsDB4) leads to the

conclusion that polymer block hydrophobicity determines the

movement of the whole construct under electrophoresis condi-

tions. Similar trends with few discrepancies were observed for

M- (lane M0 to M4) and L-extended (lane L0 to L4) materials.

However, this phenomenon merits further investigation with

ionic polymers, as the nonionic polymers used in this work do

not directly alter mobility under electrophoresis conditions.

The triblock architecture dsDNA-b-PEG-b-dsDNA (dsTB)

was prepared in an analogous ligation procedure and was also

characterized by gel electrophoresis (Fig. 2B). Not surprisingly,

the electrophoretic mobility of extended dsTB is significantly

lower than that of pristine dsDNAs due to the incorporation of

two extended DNA segments. Once more, the gel bands were

discrete, attesting to the purity of the products.

We have further characterized one of the triblock architectures

(S-dsTB) using AFM. Fig. 2C shows a representative image of

the triblock on a mica surface in fluid. Long structures with

consistent height (B1 nm) were observed, and the measured

average contour length (B550 nm) agrees well with the

calculated length (541 nm). The ligated products found with

the AFM tended to have kinks in the middle (marked in

Fig. S3, ESIw), implying the presence of a flexible PEG block

bridging the two long arms of the construct.9

Employing the method presented here, we achieved large

triblock architectures with molecular weight of up to

Fig. 1 Schematic of DBCs, the large dsDNA segments and the

enzymatic extension strategy for di- and triblock architectures. (A) Four

single-stranded DNA diblock (ssDB1-4) and triblock (ssTB) copolymers

with synthetic polymer segments. (B) Digestion of pBR322 by a DNA

restriction enzyme, Alw26I (also known as BsmAI), results in three

dsDNA segments (S, M and L, depicted as red, green and blue bands,

respectively) of the lengths shown (unit: bp). The restricted four-nucleotide

sticky ends are complementary to the 50-overhangs of corresponding

phosphorylated cDNAs (cDNA-S: CGGT-, cDNA-M: GGGA- and

cDNA-L: CTCA-). (C) In the presence of T4 DNA ligase, the mixture

of a chosen ssDB (or ssTB), the appropriate cDNA, and dsDNA segments

(S/M/L) results in extended dsDB or dsTB architectures. Asterisks (*)

denote the 50-ends of the DNAs.

Fig. 2 Agarose gel and AFM images of di- and triblock architectures extended with dsDNA segments. (A) An EtBr-stained gel image of extended

diblock architectures. Lanes are grouped according to the length of the ds extension (S, M and L). Pristine extended dsDNA control (lane 0) and

extended products dsDB1, dsDB2, dsDB3 and dsDB4 (lanes 1 to 4, respectively). (B) An EtBr-stained gel image of extended triblock architectures.

Pristine ds extended DNAs (lanes 0) and extended triblocks dsTB (lanes 1). DNA ladders are in thousands of bp in all gel images. (C) AFM image

of S-dsTB recorded in tapping mode in fluid (scalebars: 100 nm, height scale: 10 nm).

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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 2243–2245 2245

B3 million Da (lane L1 in Fig. 2B, L-dsTB), five times higher

than previously reported using another molecular biology

technique, PCR.9 Clearly, molecular biology tools are capable

of producing multiblock bio-synthetic hybrid polymers resulting

in well-defined ultra-high molecular weight products.

These techniques also allow further manipulation of the

structures thus produced. Because we adopted the large dsDNA

extensions (S, M and L) from the vector pBR322, we know its

whole sequence map. This knowledge enables the alteration of

our products with single nucleotide precision (3–4 A) with the

help of various nucleic acid processing enzymes. As a proof of

concept, we digested our S-, M- and L-extended constructs

with three different restriction endonucleases (DraI, Lgu and

Bpu10I, respectively) to cut them into still different sizes.

For both the di- and triblock architecture, digestions were

performed on the PEG hybrid polymers (dsDB2 and dsTB

with S/M/L), and agarose gel electrophoresis was again

employed to demonstrate successful scission (Fig. 3).

For the analysis of diblock architecture digestion, identical

concentrations of dsDB2 with S, M and L segments were

loaded into the gel after the enzymatic reaction (lane 4 of each

group in Fig. 3A). In the presence of the enzyme (lanes 2 and 4

of each group), three DNA bands appear in the gel, whereas

control samples without enzyme have only the single band

corresponding to the starting material (lanes 1 and 3 of each

group). In the restricted lanes (lanes 2 and 4), the two lowest

bands are digested fractions, the mobilities of which agree well

with the expected fragment sizes denoted below the gel

(Fig. 3). Analogous to the ligation results, the digested

products containing polymer segments have lower electro-

phoretic mobilities than the corresponding digested pristine

dsDNA product due to the presence of PEG. This is especially

apparent in the S group. Another notable feature is the

undigested band present in every restricted lane at the same

level as the control. Band intensity upon retention analysis of

the gel pictures revealed that there is B2% more residual

undigested material in the restricted polymeric samples than in

the pristine dsDNA lanes within the same group (see ESI.4wfor analysis of gel). This small difference suggests that the

presence of the synthetic polymers does not compromise

the activity of the endonuclease. This decrease by 2%

(i.e. 91% versus 93% restriction efficiency for S-DBs and

pristine DNA, respectively) is not a significant change or in

the error range of the analysis equipment, and the bands are

well separated so they can be purified for further applications

if necessary. The same restriction analysis was also performed

with triblock architectures containing S, M and L extensions

(Fig. 2B and Fig. S5, ESIw). A distinct result from this triblock

experiment is that a new band (the second band from the top

of lanes S4 in Fig. 2B and M4 in Fig. S5, ESIw) of partiallydigested fragment appears on the gel as well. These bands can

be interpreted as singly-cut materials in which the other

restriction site across the polymer was not affected. A plausible

explanation, taking into consideration a recently revealed

DNA polymerase mechanism,10 is that this singly-cut triblock

architecture is due to PEG-induced discontinuation of

molecular recognition between the enzyme and DNA as the

endonuclease slides along the dsDNA ‘rails’ in search of

restriction sites. However, gel electrophoresis results alone

are insufficient to shed light on this phenomenon. This

observation may nonetheless pave the way to further studies

of the mechanisms of DNA–enzyme interactions through the

combination of the variation of DBC properties, such as

polymer structure and DNA sequence, with another physical

technique like single molecule spectroscopy.

In conclusion, we have demonstrated a new strategy to

precisely prepare and alter extremely large DNA/polymer

hybrid structures. The enzymatic manipulation of complex,

non-natural DNA architectures constructed exclusively from

DNA is already known,11,12 however, polymer–DNA chimeras

have never been generated by enzymatic digestion and

ligation. Moreover, it was successfully demonstrated that

hydrophilic, hydrophobic and even thermoresponsive polymers

are well compatible with the respective enzymes. Our findings

represent a significant step forward for the facile preparation

of high precision polymers for the application in life- and

materials science.

Notes and references

1 P. W. K. Rothemund, Nature, 2006, 440, 297–302.2 N. C. Seeman, Nature, 2003, 421, 427–431.3 D. Martin, M. Klein, J. Haller, A. Adamis, E. Gragoudas,J. Miller, M. Blumenkrantz, M. Goldberg, L. Yannuzzi,D. Henninger, L. Wiegand, L. Chen, D. Drolet, S. Gill, J. Bill,B. Tomkinson, R. Bendele, D. O’Shaughnessy, D. Guyer andS. Patel, Retina–J. Ret. Vitreous Dis., 2002, 22, 143–152.

4 M. Kwak and A. Herrmann, Angew. Chem., Int. Ed., 2010, 49,8574–8587.

5 F. E. Alemdaroglu, K. Ding, R. Berger and A. Herrmann, Angew.Chem., Int. Ed., 2006, 45, 4206–4210.

6 Z. Li, Y. Zhang, P. Fullhart and C. A. Mirkin, Nano Lett., 2004, 4,1055–1058.

7 M. Safak, F. E. Alemdaroglu, Y. Li, E. Ergen and A. Herrmann,Adv. Mater., 2007, 19, 1499–1505.

8 F. E. Alemdaroglu, M. Safak, J. Wang, R. Berger andA. Herrmann, Chem. Commun., 2007, 1358–1359.

9 F. E. Alemdaroglu, W. Zhuang, L. Zoephel, J. Wang, R. Berger,J. P. Rabe and A. Herrmann, Nano Lett., 2009, 9, 3658–3662.

10 C. M. Etson, S. M. Hamdan, C. C. Richardson and A. M. vanOijen, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 1900–1905.

11 S. Keller, J. Wang, M. Chandra, R. Berger and A. Marx, J. Am.Chem. Soc., 2008, 130, 13188–13189.

12 S. H. Um, J. B. Lee, N. Park, S. Y. Kwon, C. C. Umbach andD. Luo, Nat. Mater., 2006, 5, 797–801.

Fig. 3 Restriction analysis of the extended di- and triblock architectures.

(A) Diblock architectures with S, M and L dsDNA segments.

(B) Triblock architectures with S dsDNA segment (see Fig. S6, ESIwfor M and L). The order of samples in each group: pristine dsDNA

without restriction enzyme (lane 1), its digested dsDNA with enzyme

(lane 2), and the corresponding dsDB2 (or TB for B) hybrid

architectures without (lane 3) and with (lane 4) enzyme. Unit: thousands

of bp. Restriction map of the analysis is shown below. Numbers denote

basepairs after the digestion with the corresponding endonucleases.

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