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Preparation of Stable Branched DNA Nanostructures: Process of Cooperative Self-Assembly Ashok Kumar Nayak, Sakti Kanta Rath, and Umakanta Subudhi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b00353 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 14, 2019

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The Journal of Physical Chemistry

Preparation of Stable Branched DNA Nanostructures: Process of Cooperative Self-Assembly Ashok Kumar Nayak,a,b Sakti Kanta Rath,b Umakanta Subudhia,c* a

DNA Nanotechnology & Application Laboratory, CSIR-Institute of Minerals & Materials

Technology, Bhubaneswar 751 013, India b

Department of Biotechnology, Ravenshaw University, Cuttack 753 003, India

c

Academy of Scientific & Innovative Research (AcSIR), New Delhi 110025, India

*Corresponding Author: [email protected], [email protected]

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ABSTRACT: The construction of functionalizable branched DNA (bDNA) relies on the designing of oligonucleotides and exploitation of their complementary chemistries. The stability of these structures largely depend on the hybridization specificity of the contributing oligonucleotides. However, most of the bDNA structures are not found suitable for in vivo application due to poor yield owing to uncharacterized hybridization efficiency and instability in biological fluids. In this report, our group has explored a mechanistic way for studying the hybridization pathway of genomic sequence derived oligonucleotides which are self-assembled to fabricate robust bDNA structures. The effect of change in nucleotide sequence on bDNA stability was studied by taking oligonucleotides derived from primers of different genes. Additionally, the stability of the bDNA in solutions with different pH, salts and DNaseI which mimics physiological environment was reported. It was found that genomic sequence derived oligonucleotides selfassembled in a cooperative manner to yield the designed bDNAs which are stable in physiological environment.


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INTRODUCTION The decisive control over the spatial arrangement of individual components in bottom-up synthesis of self-assembled nanostructures is a fundamental requisite in nanotechnology and synthetic biology1,2. The innate physical and chemical properties of contributing elements dictate the pathway of self-assembly and play a crucial role in structural stability and functional efficiency3. DNA, by virtue of its inherent properties like Watson-Crick base pairing, structural rigidity as well as flexibility over a nano-dimension, and ease in designing and programming, has drawn the keen interest of interdisciplinary researchers for bottom-up fabrication of convoluted nano-architectures with multi-dimensional utility4-6. Especially branched DNA (bDNA), nanostructures that conflict with conventional linear double stranded DNA, with the introduction of kink at internal regions have been evolved as tools for nanoelectronics, sensing and biomedical application7,8. DNA nanoarchitectures like DNA pyramid, triangular DNA prism, rolling circle amplified Y-DNA and tetrahedral DNA have been exploited to harness gene silencing through RNA interference (RNAi)9-12. Our group has also reported the ability of monomeric bDNA structure to regulate synergistically multiple oncogenic micro RNAs in breast cancer cells13.Despite the conception of a variety of DNA nanostructures and their functional entanglement within biomedical research, a mechanistic study about the formation of individual DNA nanostructures and the circumstances influencing structural yield and stability has not yet been fully explored14. So, a fundamental understanding about the mechanism and the chemical environment controlling the DNA selfassembly is required to improve in designing and constructing various DNA nanostructures having the potential for in vitro as well as in vivo applications. With better understanding it can be possible to overcome the errors related to structural growth, stability and target-ability of DNA nanostructures. In this scenario, it can be stated that hybridization between oligonucleotides plays 3 ACS Paragon Plus Environment


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an indispensable role in the synthesis of DNA nanostructure15. Therefore, it is necessary to examine the pathway by which these uniquely designed oligonucleotides assemble into double helix and finally lead to predesigned DNA nanostructure. In this context, it is also important to note that nucleotide sequences play a fundamental role in the hybridization behaviour of Oligonucleotides16. It has been observed that hybridization between single stranded DNA containing repetitive motifs leads to nonspecific products which result multiple intermediate species. On the contrary, oligonucleotides with random sequences codes for restrictive mode of hybridisation ensuring the desired product17,18. Moreover, inside the restrictive hybridization, cooperative

binding

of

complementary

oligonucleotides has been preferred over random binding19. Therefore, designing of oligonucleotide sequences for DNA nanostructure formation and their self-assembly pathway must be well studied. For the self-assembly among Oligonucleotides, aqueous environment is a prerequisite. The water activity in combination with different chemical stimuli like pH and ionic strength modulates the structural responsiveness of DNA nanostructures20. Usually, DNA nanostructures are self-assembled in buffers containing a sub-millimolar concentration of Na+ and Mg2+ which counteracts the repulsive force between hybridizing DNA strands21. However, a deviation from this concentration of ions may result in structural collapse or aggregation of DNA nanostructures which will eventually hamper functionality22. So, it is required to find out the optimal salt concentration and pH beyond which the structural integrity of DNA nanostructures becomes a concern. Moreover, for biomedical application the bDNA nanostructure should show structural stability in physiological pH and salt environment and also in the presence of DNA degrading enzymes. In this communication, we have investigated the self-assembly mechanism and structural stability of monomeric bDNA structures in different physiological condition. The 4 ACS Paragon Plus Environment

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bDNA structures containing oligonucleotides are derived from primer sequences of different genes (G3PDH, GPx, Catalase, SOD1, SOD2, β-actin and GR) of Rattus norvegicus since genomic sequence derived oligonucleotides are proved to be resulting stable bDNA with higher yield23. The designing of oligonucleotides is such that two oligonucleotides share either zero or 50% complementarity (Scheme 1, Table S1 and Supplementary information). After self-assembly, the resulting monomer bDNA USA-1 contains 30 bp of internal and 15 bp of external hybridizing regions with four sticky overhangs and a central bubble-like structure. The spatial arrangement of oligonucleotides has been studied computationally and found that bDNA monomer is a twodimensional planar structure (Figure S1). Additionally, the four sticky overhangs are exerted to the outer side of the bDNA and accessible for ligand binding. Once the pathway of hybridization is revealed then the effect of change in nucleotide sequences on structural stability is assessed by taking different primer sequences (SOD1 and SOD2 genes) at the internal regions and fabricating another monomeric bDNA USA-2 (oligos A, B, C, and J from Ref 23).

Scheme 1. Schematic

presentation

of

bDNA USA-1 containing oligonucleotides derived from primer sequences of different genes. To establish the generality of the approach two more bDNAs, USA-3 and USA-4 were used. bDNA USA-3 is designed by trimming the internal region (6 nucleotides from both end) of 5 ACS Paragon Plus Environment


bDNA USA-2 whereas USA-4 is an earlier reported Y-shaped bDNA synthesized using same genomic sequences. Since, the bDNA monomer through its sticky ends has the ability to perform in vivo applications, the structural stability in the physiological environment is studied. The bDNA monomers are subjected to the physiological pH range of pH 4.5 to 8.5 and stability is observed through native polyacrylamide gel electrophoresis (nPAGE), melting curve studies and circular dichroism (CD) spectroscopy. Since structural integrity of DNA is susceptible to salt ion concentration, the effect of monovalent (Na+ and K+), divalent (Mg2+ and Zn2+) and trivalent (La3+ and Ce3+) ions on bDNA monomers are studied through agarose gel electrophoresis. Finally, we tested the sensitivity of bDNA monomers to DNaseI, a DNA degrading enzyme that is present in the cellular environment through agarose gel electrophoresis.

MATERIALS AND METHODS 1. Self-Assembly Study: All the oligonucleotides mentioned in the supplementary information are purchased from Integrated DNA Technologies, Inc. and used without further purification. Self-assembly among the oligonucleotides was carried out in TAE/Mg2+ buffer that contains Tris-base (40 mM, pH 8.0), acetic acid (20 mM), EDTA (2 mM) and Mg(Ac)2 (12.5 mM). For self-assembly, contributing oligos were added in equimolar ratio (25 pmol each) with final volume of 25 µl, denatured at 95 °C and then cooled to 4 °C with ramp rate of 0.3 °C/sec using thermal cycler (Bio-Rad). The self-assembled bDNA structures are then directly used for characterization without further purification. 2. Native Polyacrylamide Gel Electrophoresis: 10% native polyacrylamide gel (acrylamide:bis acrylamide::29:1) was prepared by adding 9.422 ml milliQ water, 450 μl 50X TAE 6 ACS Paragon Plus Environment

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buffer, 7.5 ml acrylamide solution, 2.815 ml Mg(Ac)2 (0.1 M), 300 μl APS (10%) and 15 μl TEMED to make the gel volume of 22.5 ml which is sufficient for making two gels (10 cm x 10 cm). The gels were run at 4 °C for 2.5 h in a SE260 vertical electrophoresis unit (Hoeffer) at a constant voltage of 150 V by taking 1xTAE as running buffer. After electrophoresis the gels were stained with ethidium bromide solution (0.5 μg/ml) for 45 min and then documented in FluroChem E system (Cell Biosciences). 3. Melting Curve Analysis: For melting curve analysis 5 μl of self-assembled bDNA was added with 0.5 μl of SYBR Green (100x, Thermo Fisher) and final volume was made to 25 μl with 1x TAEM buffer. The sample was mixed by slight tapping and then vortexing followed by incubation for 10 min at room temperature. Then the sample was heated to 95 °C for 5 min and then cooled to 4 °C within 30 min in an Eppendorf Master Cycler. The melting curve was obtained as a relative change in luminescence (-dl/dT) with temperature (T) by the built-in software of master cycler. 4. bDNA Stability in Presence of Cations: For this experiment the chloride salts of Na, K, Mg, Zn, La, and Ce were purchased from Sigma Aldrich. 5 μl of bDNA (1μM) was incubated with different concentration of cations at 37 °C in water bath for 1 h. Then the sample was gently mixed with pipette before running in 1.5% agarose gel for 20 min. For melting curve analysis of above samples, 0.5 μl of SYBR Green (100x, Thermo Fisher) was added to each tube containing bDNA and cations followed by incubation for 10 min at room temperature and then put in Eppendorf Master Cycler. 5. Circular Dichroism Study: The CD spectra of self-assembled bDNA structures were measured using chirascan spectrophotometer (Applied Photophysics). The spectra were recorded with scan speed of 60 nm/sec, bandwidth of 1 nm and a time per point of 0.5 sec. All measurements 7 ACS Paragon Plus Environment


were done at 25 °C between wavelength of 320 nm and 200 nm by using a quartz cuvette of 1 mm path length. Three spectra per sample were averaged for getting the final spectrum and final spectra were corrected for hybridizing buffer background. 6. DNaseI Stability Study: The stability of bDNA structure in presence of DNaseI was conducted by taking 12.5 μl of bDNA and its di-, tri-oligo complexes from 2 μM self-assembled reaction mixture and then digested with 0.2 unit of DNaseI. After slight mixing the samples were incubated at 37 °C in water bath (ORBIT) for a period of 1, 2, 5, 10, 15, 20 and 30 min. The digested products were then analyzed by agarose gel electrophoresis (1.5%, 100 V for 20 min). The densitometric analysis of DNA bands in agarose gel with their respective time intervals were conducted through ImageJ software to obtain band intensity values which can graphically testify to the stability of corresponding structures in DNaseI.

RESULTS AND DISCUSSION The proposed bDNA monomeric structure, USA-1, is the result of self-assembly among four oligonucleotides. The designing of oligonucleotides was so specific that non-complementary oligonucleotides never interacted with each other to form any spurious product, rather maintained their identity as a single species in the reaction mixture (Figure S2). It was observed that oligonucleotide USA-1a and USA-1c, USA-1a and USA-1d or USA-1b and USA-1d did not hybridize during thermal annealing. Hence, these unassembled oligonucleotides remained as an individual entity in the self-assembly reaction mixture which could be seen in the nPAGE. This result corroborated with our earlier observation23. When two oligonucleotides sharing complementary nucleotides were self-assembled at an equimolar ratio (1µM), they formed the 8 ACS Paragon Plus Environment

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respective di-oligo complex which could be confirmed by the presence of a predominant band with higher intensity and absence of unused oligonucleotides at the base of the nPAGE gel as compared to other molar ratios (Figure S3). Hence, equimolar ratio of oligos was used throughout this work to obtain the desired product. The formation of bDNA USA-1 was observed with a predominant band in 10% nPAGE. However, a stepwise decrease in electrophoretic mobility was seen between di-, trioligo complex and bDNA USA-1 due to sequential addition of contributing oligos and resulting structural configuration (Figure S4). The difference observed in electrophoretic mobility among di-oligo complex (USA-1ab, USA-1bc and USA-1cd) was in accord to the different overall structural configuration. The di-oligo complex USA-1bc contains an internal bubble of 30 nucleotides. Such bubbles are more flexible than corresponding double stranded DNA24. Thus, USA-1bc had higher electrophoretic mobility in comparison to other di-oligo complexes. However, the difference in electrophoretic mobility between tri-oligo and monomeric bDNA was due to the increase in molecular weight. To provide insight into the mechanism of self-assembly among oligos to form bDNA USA1, melting curve analysis was conducted. bDNA USA-1consists of four hybridizing regions, two with 30 bp regions in the inner position (USA-1ab and USA-1cd) and the other two with 15 bp regions in the outer position (USA-1bc). Therefore, it is first required to study the annealing behaviour of those hybridizing regions. For this, the oligo complexes were first stained with SYBR Green-I (SG) and then denatured through a temperature dependent reaction. The measurements were based on the fact that SG fluoresces more with hybridized oligonucleotides than free oligos. Figure 1 depicted the melting behaviour of di-oligo, tri-oligo and monomeric bDNA showing different peak values which corresponded to their melting temperature (Tm). The di-oligo complex USA-1ab and USA-1cd showed Tm values of 83.5°C and 81.5°C respectively whereas USA-1bc 9 ACS Paragon Plus Environment


showed Tm at 68.6°C (Figure 1A). The higher Tm values of USA-1ab and USA-1cd is due to the 30 bp continuous double stranded region. On the contrary, the 30 bp region of USA-1bc is divided into two 15 bp regions with a bubble in between. Such internal bubbles cause an entropic penalty leading to a decrease in Tm values25. This observation is in line with the formation of heterotropic DNA nanostructures reported earlier19. The formation of bDNA USA-1 proceeded through the occurrence of two key intermediates USA-1ab and USA-1-cd which provides a stage to control the yield of the final product. The melting curve of tri-oligo USA-1-bcd showed two Tm values, 64.5 °C and 82 °C, which corresponds to the two different hybridization regions of USA-1bc and USA-1cd, respectively. The decrease in Tm values of USA-1bc regions from individual parts probably due to the structural load on one side of the internal bubble. A similar phenomenon was observed for USA-1abc with decreased Tm value of 63.3°C for USA-1bc region and 83.3°C for USA-1ab region. Moreover, in bDNA USA-1 the stability of USA-1bc region slightly decreased up to 60.7 °C in comparison to USA-1abc and USA-1bcd. It has been proved that the integrity of a DNA nanostructure is based on the stability of weak hybridization regions26. In this regard, bDNA USA1 nanostructure is highly stable with lowest Tm value of ~61 °C, which is higher than physiological temperature and supports for its in vivo applications.


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Figure 1. Melting

curve analysis of (A) di-

oligos USA-1ab, USA-1bc, USA-1cd, tri-oligos complexes USA-1abc and USA-1bcd, bDNAUSA-1 and (B) di-oligos USA-2ab, USA-2bc, USA-2cd, tri-oligos complexes USA-2abc and USA-2bcd and bDNA-USA-2. In both the structures the 30 bp internal regions of USA-1 and USA-2ab showed higher Tm in comparison to the 15 bp duplex regions. To evaluate the effect of nucleotide sequences on the stability of bDNA monomer, we used earlier reported bDNA monomer in which the internal hybridizing sequences of bDNA USA-1 have been replaced with the nucleotide sequences derived from SOD1 and SOD2 genes. The resulting bDNA USA-2 and its intermediate complexes were then denatured in presence of SG and corresponding Tm values were analysed (Figure 1B). The internal hybridizing regions of USA2ab and USA-2cd have Tm values of 82.2 °C and 79 °C respectively. The slight decrease in Tm of internal regions of bDNA USA-2 in comparison to that of bDNA USA-1 is due to the decrease in GC content. Table S2 depicts the relationship between GC content of hybridizing regions of both 11 ACS Paragon Plus Environment


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the bDNAs with their stability in terms of Tm values. Since the nucleotide sequences of external hybridizing regions are kept constant there is hardly any change in Tm values. From this observation, it can be postulated that primer sequences with higher GC content should be utilized for bDNA fabrication. Though there is slight difference in Tm values, both the bDNAs are stable at physiological temperature. DNA nanostructures designed for the biomedical application needs to be stable in different extracellular and intracellular environment within a pH range of 4.5 to 8.527. In this context, the stability of bDNA nanostructures (bDNA USA-1 to USA-4) in different pH environment can be studied

by

two

methods.

First,

by

performing

self-assembly

at

different

pH

Figure 2. Characterizing the stability of bDNA nanostructures in different pH through A) densitometric analysis of 10 % nPAGE and B) melting curve analysis. 12 ACS Paragon Plus Environment

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or, in the second method, incubating self-assembled DNA nanostructures in different pH. Here, we followed the first method and self-assembled the bDNA structures in a pH range of 4.5 to 8.5 and evaluated the effect of ionic environment on structural yield and stability. When electrophoresed, the band intensities of bDNA USA-1 remained constant within the pH 5.5 to 8.5 (Figure 2A and Figure S5A). However, acidic pH of 4.5 affected the formation of bDNA USA-1 and observed through decreased band intensity. Similar effect was also observed when other bDNA structures, USA-2, USA-3 and USA-4 were self-assembled in pH range of 4.5 to 8.5 (Figure 2A and Figure S6). bDNA USA-3 showed lowest stability in acidic pH where as bDNA USA-1 was found to be the most stable among these four bDNA structures. This decreased structural stability is due to the depurination occurred by acid catalyzed hydrolysis of DNA at acidic pH28. Complex DNA nanostructures like DNA origami was also reported to be sensitive to decrease in pH20.This phenomenon can also be supported through melting curve analysis and CD spectroscopy (Figure 2B and Figure S5B). When denatured at different pH, bDNA-USA-1 showed unchanged Tm value through pH 5.5 to 8.5. However, a decrease in Tm value at pH 4.5 was observed. The CD spectra of bDNA USA-1 assembled in different pH exhibited positive peaks at 275 nm and 220 nm and a negative peak at 250 nm which is the signature spectra of right handed B-DNA conformation29.However, it could be pointed out that the peak intensities remained constant through pH 5.5 to 8.5, but slightly decreased at pH 4.5 which was due to decrease in yield and stability. Similar effect of acidic pH on CD spectra of DNA triplex was monitored 30. Since the bDNA USA-1 showed its stability in physiological pH range, this advocates its potential in in vivo applications.



Figure

3.

Effect

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monovalent (Na+ and

of

K+), divalent (Mg2+ and Zn2+) and trivalent (La3+ and Ce3+) cations on structural stability of bDNA USA-1. The structure remained stable even at 500 mM concentration of Na+ and K+ but condensation occurred at 500 mM of Mg2+, 50 mM of Zn2+ and 10 mM of La3+ and Ce3+. The concentration of salts is mentioned on top of each lane and the salt type is mentioned below each gel. DNA, being a polyanionic structure in physiological conditions, presents conformational behaviour and functional dynamics largely depending on the local availability of positively charged species like metal ions31. Moreover, the formation of multi-stranded DNA structure formation requires the presence of counter-cations to suppress phosphate-phosphate repulsion which arises due to an increase in the localized concentration of nucleic acids29,32. So to understand the effect of monovalent (Na+ and K+), divalent (Mg2+ and Zn2+) and trivalent (La3+ and Ce3+) ions 14 ACS Paragon Plus Environment

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on bDNA nanostructures, bDNA USA-1, USA-2, USA-3 and USA-4 were incubated with different concentrations of chloride salt of Na+, K+, Mg2+, Zn2+, La3+, and Ce3+ at 37°C for 1 h. When bDNAs were added with Na+ or K+, the bDNA structures sustained structural integrity even at 500 mM of salt which could be assessed from constancy in band intensities in agarose gel (Figure 3 and Figure S7). However, a decrease in band intensity and retarded mobility was observed at 100 mM and 500 mM of MgCl2. Similar phenomena were observed for ZnCl2 at lower concentration (50 mM) than MgCl2. Such retardation in electrophoretic mobility was due to condensation of DNA33. Among the divalent metal ions, Zn2+ has the strongest binding specificity for the phosphate backbone and nitrogen bases of DNA at pH 7.4 thereby neutralizing the negative charge and increase the base stacking34. This increase in base stacking renders inefficient binding of intercalating molecules like ethidium bromide. Thus, there is a decrease in band intensities observed with increase in Zn2+ concentration. This structural transition also leads to the condensation of bDNA. On the other hand, addition of 10 mM chloride salts of La or Ce caused a similar bDNA condensation observed through band shifting in agarose gel. Lanthanides mediated DNA condensation and structural transition has been reported29,35,36. Moreover, it has been proved that monovalent cations stabilize multiplex DNA structures by reducing electrostatic repulsion thereby increasing melting temperature34.



Figure 4. Melting curve analysis of bDNA USA-1 titrated with chloride salts of (A) Na, (B) K, (C) Mg, (D) Zn, (E) La and (F) Ce. Higher concentration of Na and K stabilized the bDNA which is shown by increased Tm value. DNA condensation occurred when 100 mM of chloride salts of Mg or 10 mM of Zn, La or Ce was added which can be seen through the unusual spectra obtained during melting curve analysis. When bDNA USA-1 was denatured in presence of Na+ and K+ the Tm increased with increase in cation concentration from 1 mM to 500 mM (Figure 4). But for Mg2+, the Tm value remained constant throughout the concentration from 1 mM to 500 mM. However, a decrease in 16 ACS Paragon Plus Environment

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peak intensity was observed after 10 mM which was due to compaction of DNA. Mg2+ has binding affinity only for the phosphate backbone in DNA, hence showed lower tendency towards DNA condensation. On the contrary, divalent cations like Zn2+ and trivalent cations usually binds to both the phosphate backbone and nitrogen bases in DNA34,37. This leads to DNA condensation even at lower cation concentration in comparison to monovalent cations and Mg2+. Moreover, such DNA condensation does not allow efficient binding of intercalating dyes like SG. So, Tm analysis of Zn2+ induced condensed bDNA USA-1 produced unusual peaks. Similar observation was noticed when bDNA USA-1 titrated with La3+ or Ce3+ was denatured in presence of SG. Summarising, the bDNA USA-1 was stable at higher concentration of Na+ and K+ and sub-millimolar concentration (10 mM) of Mg2+ and Zn2+. However, the condensed bDNA resulted with Zn and trivalent cations can have other advantages like ease in transfection. Condensed si-RNA by Zn2+ as a reliable method for carrier free gene delivery has already been reported34.

Figure

DNaseI

5.

Stability study of bDNA USA-1 through agarose gel electrophoresis after incubation at 37°C for 0 to 30 min. bDNA USA-1 showed highest stability as compared to the respective di-and tri-oligo complexes. 17 ACS Paragon Plus Environment


Further, we examined the stability of bDNA structures against nonspecific enzymatic degradation where the structural configuration of DNA matter the most over sequence composition. For this, bDNA USA-1 was incubated with DNaseI, an endonuclease that cleaves DNA. The experiment was conducted at physiological pH 7.4 and temperature of 37 °C with incubation period upto 30 min and the stability of bDNA USA-1 in comparison to its intermediate di-and tri-oligo complexes were accessed through agarose gel electrophoresis (Figure S8). Figure 5 depicted the sensitivity of di-oligo complexes USA-1ab and USA-1bc, tri oligo complex USA1bcd and bDNA USA-1 towards DNaseI activity. 50% of USA-1ab, USA-1bc got degraded by DNaseI activity within 2 min whereas half of USA-1bcd was found to be degraded in 11 min. However, bDNA USA-1 showed highest resistance to DNaseI mediated degradation having 50% structural integrity up to 18.5 min. Moreover, bDNA USA-1 was more stable structure than USA2, USA-3 and USA-4 (Figure S9). This increase in structural resistance to digestion is attributed by the increase in complexity of both local and overall helix geometry. However, this bDNA showed higher resistance to digestion by DNaseI than earlier reported DNA nanostructure38. Moreover, bDNA USA-1 showed greater stability in comparison to its contributing intermediate DNA complexes at a DNaseI concentration which is large excess as compared to reported serum level38,39. This experiment again supported the potential of bDNA USA-1 for in vivo applications.

CONCLUSIONS Genomic sequence derived monomeric bDNA nanostructures have been emerged as potent therapeutic modalities. In this work, the mechanism of self-assembly among oligonucleotides to form bDNA nanostructure has been revealed. We observed that nucleotide sequences derived from primers of different genes codes for restrictive mode of hybridization which led to formation of 18 ACS Paragon Plus Environment

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required single molecular species. Moreover, the co-operative mode of binding between intermediate species provides a platform for structural modification. The resulting bDNA structure USA-1 has shown structural resistance to buffers with physiological pH range of pH 4.5 to 8.5. bDNA USA-1 also maintains its structural stability at physiological concentration of mono and divalent cations. However, at higher concentration (≥ 50 mM Zn2+, ≥ 10 mM La3+ and Ce3+) bDNA gets condensed and this condensed bDNA structure can find importance in carrier-free gene delivery applications. The multi-stranded bDNA structure is also resistant to DNaseI mediated degradation due to different spatial orientation and localized nucleic acid concentration in comparison to duplex DNA. Since the designed bDNA USA-1 has demonstrated structural stability at physiological environment, this advocates for its use in in vivo applications. ACKNOWLEDGMENT This work was supported by the Council of Scientific and Industrial Research (CSIR), Government of India with EMPOWER OLP-19 and YSP-05. AKN acknowledges CSIR for providing Senior Research Fellowship. Supporting Information Available: Details of oligonucleotide sequences (Table S1), relationship between GC content and Tmvalue (Table S2), characterization of oligo complexes and their stability (Figure S1-Figure S9) has been provided in this file. This material is available free of charge via the Internet at http://pubs.acs.org. CONFLICTS OF INTEREST There are no conflicts to declare. REFERENCES 19 ACS Paragon Plus Environment


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Schematic presentation of self-assembled DNA nanostructures and their stability in presence of different pH and metal ion concentrations. It was found that genomic sequence derived oligonucleotides self-assembled in a cooperative manner to yield the designed bDNA structures which are stable in physiological environment and can be applied for in vivo applications.

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Preparation of Stable Branched DNA ... - ACS Publications

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