Probing DNA's Interstrand Orientation with Gold Nanoparticles

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Probing DNA’s Interstrand Orientation with Gold Nanoparticles Xue Bai, Jinjing Wu, Xiaogang Han, and Zhaoxiang Deng* CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China

bS Supporting Information ABSTRACT: The interstrand orientation of a DNA duplex plays a pivotal role in its biological and chemical functions. Therefore, developing an efficient way to determine (control and monitor) the parallel or antiparallel conformation of a DNA duplex is of great significance, which, however, remains a big challenge under some circumstances. In this work, we demonstrate that gold nanoparticles tagged on DNA are especially useful in trapping and detecting a special interstrand orientation of a DNA double helix, based on inherent electrostatic and steric repulsions between nanoparticles which will affect their self-assembly into a large structure. More importantly, some of the conformations revealed by the gold nanoparticle assay may even not be thermodynamically preferred and thus will be hard to detect using currently available methods. This simple, straightforward, and efficient methodology capable of dictating and probing a special DNA duplex structure provides a useful tool for conformational analyses and functional explorations of biomolecules as well as biophysical and nanobiomedical research.

D

NA’s potential in adopting alternative conformations is of great importance due to its central role in life sciences. In contrast to its antiparallel (AP) conformation in a canonical Watson
Crick model, DNA can also take a parallel-stranded (PS) duplex structure.1
5 However, a big challenge is how to find a suitable way to directly interact with the formation of a DNA duplex and thus to achieve an efficient control on its conformational variants (PS or AP). Such a control as well as the detection of the interstrand orientations of a DNA duplex is long expected for their structural and functional elucidations in a biological or chemical context.1
5 Here, we report on the use of gold nanoparticles (AuNPs) to probe and control the formation of a DNA duplex, whereby its interstrand orientation can be virtually “set” and “seen”. Our findings may further enable the investigations toward structure
function relativity of a special DNA conformation in vitro and in vivo. To probe the interstrand orientation within a DNA duplex, F€orster resonance energy transfer (FRET), pyrene excimer fluorescence and various other spectroscopic methods are often utilized.6
11 Unfortunately, these “passive” techniques are unable to harness the structure of a DNA molecule. The past decade has witnessed various important applications of AuNPs including colorimetric sensors,12
20 DNA programmed nanoassembly,21
28 gene or drug delivery,29
33 colloidal crystallizations,34
37 and control over the helicities of DNA nanotubes by interparticle hindrance.38 Recently, it has also been found that redundant DNA overhangs could affect the stability of AuNP aggregates cross-linked by DNA hybridizations, due possibly to the increased steric hindrance within the assemblies.39 Apart from these examples, we herein present an important application of AuNPs from a previously unexplored angle to sensitively probe and reliably control the conformational PS or AP interstrand orientations of DNA double helices. r 2011 American Chemical Society

’ EXPERIMENTAL SECTION Materials. HAuCl4 3 4H2O, formamide, and sodium acetate were purchased from Sinopharm Chemical Inc. (Shanghai, China). Sodium chloride, boric acid, ethylenediamine tetraacetic acid disodium salt (EDTA 3 Na2), and DNA oligonucleotides were obtained from Sangon Bioengineering Technology and Services Co. Ltd. (Shanghai, China) in purified forms (modified oligos and unmodified oligos were purified by HPLC and PAGE, respectively). The purities of the DNA oligonucleotides were further checked by denaturing PAGE (8 M urea) before use. Agarose, acrylamide, bis-acrylamide, and tris(hydroxymethyl) aminomethane (Tris) were obtained from Bio Basic Inc. (BBI, Canada). Sodium citrate tribasic dihydrate and stains-all were from Sigma. Gold nanoparticles (AuNPs, 13 nm) were synthesized following the procedures described in the literature.40,41 The sequences of the DNA oligonucleotides for the preparations of DNA conjugated AuNPs (named as Au1
8 for short) are listed as below (note that one AuNP was conjugated with multiple DNA strands): Au1, AuNP-S-50 TTTTTATTAAATATA 30 ; Au2, 50 AAAAATAATTTATAT 30 -S-AuNP; Au3, AuNPS-50 AAAAATAATTTATAT 30 ; Au4, AuNP-S-50 AATTAAAAAAATTAA 30 ; Au5, AuNP-S-50 TTAATTTTTTTAATT 30 ; Au6, 50 TTAATTTTTTTAATT 30 -S-AuNP; Au7, AuNP-S-50 GAGAGAGAGAGAGAGAGAGAGAGAG 30 ; Au8, 50 GAGAGAGAGAGAGAGAGAGAGAGAG 30 -S-AuNP. Preparations of Au1
6. 50 (Au1, 3, 4, and 5) or 30 (Au2 and Au6) thiolated DNA oligonucleotide was combined with AuNPs Received: April 17, 2011 Accepted: May 31, 2011 Published: May 31, 2011 5067

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Analytical Chemistry (13 nm diameter) at a 100:1 molar ratio in 0.5 TBE (Tris, 44.5 mM; EDTA, 1 mM; boric acid, 44.5 mM, pH 8.0) buffer with final concentrations of 4 μM and 40 nM for DNA and AuNPs, respectively. NaCl was gradually introduced to the above solution within 24 h to reach a final concentration of 0.15 M. The solution was centrifuged at 9560g for 15 min, and the resulting precipitate of AuNPs was collected and redispersed in 0.5 TBE supplemented with 0.1 M NaCl. Preparations of Au7 and Au8. 50 (Au7) or 30 (Au8) thiolated DNA and AuNPs were mixed at a 20:1 molar ratio (final concentrations, 0.4 μM and 20 nM for DNA and AuNPs, respectively) in 0.5 TBE buffer supplemented with 50% (v/v) formamide serving as a chemical denaturant to prevent the poly(GA) sequence from forming homodimers, which once formed would decrease the hybridizability of the resulting DNA
AuNP conjugates. In addition, the ratios between DNA and AuNPs were purposely decreased to 20:1 with an intention to reduce the chance of basepairing between adjacent DNA strands sitting on the same gold nanoparticle. After an increase in the concentration of NaCl to 0.1 M within 24 h, the AuNPs were isolated as precipitates via centrifugation (9560g, 15 min) and redispersed in 0.5 TBE. Hybridizations of DNA-Capped AuNPs. Two parts of DNA
AuNP conjugates (i.e., Au1 þ Au2, Au1 þ Au3, Au4 þ Au5, Au4 þ Au6, or Au7 þ Au8) were combined at 1:1 molar ratio (based on optical absorbance of AuNPs at 520 nm) in 0.5 TBE supplemented with different concentrations of NaCl (see Table S1 in the Supporting Information) at 4 °C. Hybridization times and NaCl concentrations were varied according to the requirements of different characterization techniques (see Table S1 in the Supporting Information). Large aggregates (formed in the presence of a high concentration of NaCl with significant electrostatic screening between AuNPs) would lead to sedimentations of AuNPs, which could cause unstable readouts during DLS and optical absorbance measurements. Accordingly, lower NaCl concentrations or shorter hybridization times were used in circumstances where small and thus stably dispersed AuNP aggregates were preferred. Characterization Methods. Agarose and polyacrylamide gel electrophoresis, transmission electron microscopy (TEM), dynamic light scattering (DLS), and absorbance, fluorescence, and circular dichromism (CD) spectroscopies were employed for sample characterizations. See the experimental section in the Supporting Information for details.

’ RESULTS AND DISCUSSION Figure 1 shows the basic theory of our method. From an energy point of view, it is logically understandable that forming a stable AuNP-tagged DNA duplex requires that electrostatic repulsions as well as steric hindrances between the negatively charged AuNP tags being minimized (i.e., two AuNPs tend to stay far away from each other, Figure 1a). Most importantly, it is now possible to “check out” one desired interstrand orientation when two conformational alternatives (AP and PS) coexist (Figure 1b). This scheme is also valid in probing the interstrand orientation of a DNA homoduplex (as a result of dimerization of the same DNA strand, Figure 1c), where FRET based strategy does not function well (i.e., only part of the formed DNA duplexes will bear correct FRET dye pairs, and the use of a pyrene-excimer fluorescence based strategy usually has to be considered). To demonstrate the validity of our strategy, we first chose to work on a well-investigated type of PS DNA, whose sequence (composed of only “A” and “T” bases, Figure 2a) was designed to

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Figure 1. Schematic representation of a gold nanoparticle based strategy capable of probing and controlling the interstrand orientations of a DNA duplex. (a) A simplified model showing distinctively different Coulombic and steric repulsions between two gold nanoparticles located at the same and opposite termini of a DNA duplex. (b) With selective variation of the labeling positions of the nanoparticle tags on two DNA strands whose base sequences are centrosymmetric, it is possible to “check out” either a parallel or an antiparallel-stranded DNA duplex. (c) The gold nanoparticle probe is similarly effective in finding out the correct interstrand orientation of a DNA homoduplex. In an actual case, one gold nanoparticle was conjugated with multiple DNA strands and hybridizations between them resulted in nanoparticle aggregates that could be conveniently judged by naked eyes or other characterization techniques.

rule out the possibility of forming an AP duplex. Nondenaturing polyacrylamide gel electrophoresis (PAGE) evidenced the hybridizations between corresponding DNA sequences (Figure S1 in the Supporting Information). In Figure 2a, strong Coulombic and/or steric repulsions between neighboring AuNPs determined that only a HeteroPS-1 structure (Au1 þ Au2) would be valid, which led to the formation of aggregated AuNPs (note, multiple DNA strands were modified on the same AuNP, and therefore hybridizations between different AuNPs resulted in nanoparticle aggregates). Dynamic light scattering (DLS) data in Figure 2b (blue curve) (also see Figures S2
S4 in the Supporting Information) detailed the size-distribution (numberweighted) of the AuNP aggregates (HeteroPS-1). In the case of “Au1 þ Au3”, the HeteroPS-2 duplex (AuNP conjugated) could not be stabilized due to strong electrostatic/steric repulsions between the AuNPs tethered at the same end of a DNA duplex, and as a result, no aggregation of AuNPs happened (Figure 2b, red curve). TEM (Figure 2c and Figures S5
S9 in the Supporting Information) provided further evidence for the AuNP-dictated formation of a parallel-stranded duplex (HeteroPS-1). In addition to the above characterizations, agarose gel electrophoresis appeared to be a very sensitive technique that could be employed to monitor the degree of AuNP aggregations: slowly moving or smeared gel bands corresponded to nanoparticle aggregates.20,23,42,43 Besides, there was clearly a red-shift of the surface plasmon resonance (SPR) peak on the visible absorbance spectrum of the aggregated “Au1 þ Au2” sample (Figure S10 in the Supporting Information). 5068

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Analytical Chemistry

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Figure 2. PS-DNA was chosen to demonstrate the determination of its duplex orientation by AuNPs. (a) Schematic drawing demonstrating the role of gold nanoparticles. (b) DLS data (see Figure S4 in the Supporting Information for a temperature-dependent stability of the AuNP aggregates as measured by DLS) showing the size-distributions of aggregated (HeteroPS-1) as well as dispersed (HeteroPS-2) AuNPs. (c) A typical TEM image of the HeteroPS-1 sample (see also Figures S5
S9 in the Supporting Information for TEM images of all related samples). (d) Agarose gel separation verifying the hybridization scheme in part a. Figure S1 in the Supporting Information provides a nondenaturing PAGE showing the hybridizations between untagged DNA strands. See Figures S10 and S20 in the Supporting Information for optical absorbance-based characterizations of the samples.

By choosing two DNA strands (see Figure S11 in the Supporting Information for a nondenaturing PAGE showing their hybridizations), each of which featured a centrosymmetric base sequence containing only A and T (see Figure 1b and Figure 3a for details), we further demonstrated that AuNPs were able to force DNA to selectively adopt either an AP or a PS conformation each time. As depicted in Figure 3a, the AuNPs tagged on the 50 termini of the two cDNA strands prefer an antiparallel structure to achieve minimized repulsions between two AuNPs. On the contrary, the above DNA strands with the AuNPs tagged on a 50 and a 30 end will take a parallel-stranded form of a double helix. Evidences from DLS (Figure 3b and Figures S12
S14 in the Supporting Information), TEM (Figure 3c, see also Figures S15
S19 in the Supporting Information for TEM images of all related samples), gel electrophoresis (Figure 3d), and optical absorbances (Figures S20 and S21 in the Supporting Information) confirmed these predictions. The above results stimulated us to find out the prevalent interstrand orientation (PS or AP) of the DNA duplex as shown in Figure 3a when the AuNP tags were absent. We employed an FRET-based nondenaturing PAGE assay44 to gain such information (Figure 3e). It was observed that when an FAM fluorophore and a BHQ-1 quencher were decorated on a 50 and a 30 end of the two DNA complements (Table S2 in the Supporting Information), respectively, fluorescence emission of the as-formed duplex almost disappeared (lane 5 in the fluorescent gel shown in Figure 3e). With the assumption that antiparallel orientation was the only dominant conformation of as-formed DNA duplex, it was understandable that FAM and BHQ-1 would be located on

Figure 3. Gold nanoparticles were employed to control and monitor the formation of either a parallel or an antiparallel-stranded DNA duplex. (a) Schematic of the experiments. (b
d) DLS (see also Figures S12
S14 in the Supporting Information), TEM (corresponding to the HeteroAP sample, HeteroPS was similar, see Figures S15
S19 in the Supporting Information), and agarose gel separation based characterizations. (e) FRET-based gel retardation assay. (f, g) DLS and FRET based melting profiles of as formed DNA duplexes either trapped inside AuNP aggregates (f) or labeled with fluorescent and quencher dyes (g, AuNP-free). Evidence from optical absorbance was given in Figures S20 and S21 in the Supporting Information. NaCl concentration was decreased 2.5 times (Table S1 in the Supporting Information) in the DLS sample compared to TEM and electrophoresis to avoid settling-down of large AuNP aggregates during measurements. Gel images in part e were based on a 20% nondenaturing PAGE, and the left and middle pictures in part e corresponded to dyestained (stains-all) and fluorescent gels, respectively. The right side of part e was a superimposed image. In part g, the DNA combination used for the melting experiment was identical to lane 5 of the PAGE gel. Figure S11 in the Supporting Information provided a nondenaturing PAGE showing the formation of DNA duplexes between unlabeled DNA strands.

the same side of the as-formed AP-duplex, leading to an efficient fluorescence quenching (lane 5 in Figure 3e). In contrast, when 5069

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Analytical Chemistry FAM and BHQ-1 were at the opposite sides of the AP-duplex, significant fluorescence was retained (Figure 3e, lane 7). We further employed DLS and FRET based fluorescence spectroscopy to obtain corresponding melting curves of asformed DNA duplexes. Figure 3f indicated that the antiparallel stranded poly (A,T) duplex was thermodynamically more favorable than its parallel-stranded counterpart with a melting point difference of 13.5 °C (22.7 and 36.2 °C for the PS and AP duplexes, respectively). Note that the thermal melting profile of such a parallel-stranded duplex would be very challenging to obtain without the help of AuNPs, due to the overwhelming thermodynamic “stability” of its antiparallel counterpart.5 In the absence of the AuNP tags, a FRET-based DNA melting experiment (Figure 3g, see the Supporting Information for experimental details) of the DNA duplex (bearing a 50 -fluorophore and a 30 quencher on two separate DNA strands) showed a single thermal transition from a “dark” state to a “bright” state of the fluorophore modifier, indicating that an antiparallel duplex was the major product, consistent with the FRET-PAGE results (Figure 3e). These experiments clearly evidenced that AuNPs did an excellent job in “freezing” a less stable parallel-stranded DNA structure (refer to the AuNP-free system in Figure 3g, where AP conformation dominated), which would be otherwise very difficult (if not impossible) to achieve.5 Circular dichromism (CD) absorbance spectroscopy is a wellknown technique adept at resolving different chiral conformations including DNA double helices. Regarding the AP or PS orientations of a DNA duplex, CD relies on a preknowledge of the spectral signatures of a specific structure and therefore is not as straightforward as the AuNP based strategy. Besides its incapability of controlling a special DNA structure, CD measurements also require relatively high concentrations (micromolar or above) and large sample volumes (hundreds of microliters typically) of DNA strands to achieve a satisfactory sensitivity and suffer from interferences due to the coexisting of other light absorbers and scatters. These problems do not exist for our AuNP based method thanks to its high sensitivity and the availability of multiple detection options. Figure 4a shows the CD spectra of two DNA duplexes in antiparallel and parallel-stranded orientations. It is clearly visible that the AP structure had an upward (positive) CD peak at 282 nm, while a PS conformation exhibited a relatively flat and much lower signal around this wavelength. In the case of the symmetric sequences in Figure 3a, the CD data in Figure 4b unambiguously proved that, when the DNA strands were not labeled by AuNPs (thiol tags still existed), an AP structure (in contrast to a PS structure) prevailed in both cases with characteristic CD peaks at 283 nm. The CD experiments for the AuNP-free samples agreed well with the FRET data in Figure 3e and the literature report.1
5 While attempts to obtain CD signals of the DNA duplexes under the control of AuNPs were very challenging due to the absorbance interferences from AuNPs and unpaired DNA single strands (see Figure S27 in the Supporting Information), our experiments (see Figure 4c,d) constantly revealed a resolvable upward CD peak around 286
287 nm for the AP sample in the presence of AuNPs. As expected, the PS structure in Figure 4c did not show such a peak. In addition, there were not such features on the CD curves where no hybridizations happened between the gold nanoparticles (Figure S27 in the Supporting Information). The CD data further evidenced the efficiency of the AuNP based strategy in controlling and probing the AP and PS orientations of a DNA duplex.

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Figure 4. Circular dichromism spectra of DNA duplexes formed in the absence (a, b) and presence (c, d) of AuNP tags. In part a, a PS structure was formed between two DNA strands: 50 TTTTTATTAAATATA30 and 50 AAAAATAATTTATAT30 , while an AP structure was formed between 50 TTTTTATTAAATATA30 and 50 TATATTTAATAAAAA30 . In part b, the CD curves indicated that the AP and PS combinations as shown in Figure 3a exclusively formed AP structures in the absence of AuNPs (the “PS” structure was not really formed due to the higher stability of the AP structure). Part d was a differential CD spectrum between the AP and PS duplexes in part c trapped by AuNPs. In parts c and d, despite the background interferences from unpaired DNA strands and AuNPs (such as the positive overshoots around 264 nm, also see Figure S27 in the Supporting Information), the curve in part c for the AP structure and the differential spectrum in part d exhibited an upward peak around 286
287 nm. Such a peak was very close to the CD maxima around 282
283 nm for the AP structure in parts a and b, in sharp contrast to the PS structure in part c. The scattered data points in parts c and d were experimental data, and the solid lines were smoothed spectra by adjacent averaging (five points).

Different from the poly (A,T) sequences, it has been reported that a DNA strand containing multiple GA repeats tends to dimerize into a PS homoduplex.11,45
48 Since the GA system was not as clear and well-characterized as the AT duplex, we were curious to see if our methodology could be generalized to predict this interesting structure. The DNA strand we used here contained 12 GA repeats plus one extra G at its 30 end (i.e., (GA)12G) (see Figure 5a). Nondenaturing PAGE verified the dimerization of this sequence (Figure S22 in the Supporting Information). To further probe the basepairing direction within the [(GA)12G]2 dimer, AuNP was attached to the 50 or 30 end of this strand (note that only one side, 50 or 30 , of a DNA strand would be linked with an AuNP, see Figure 5a for details). The data in Figure 5 showed that gold nanoparticles effectively aggregated when Au7 and Au8 were mixed up, while either Au7 or Au8 itself did not aggregate via the only possible antiparallel hybridizations (Figure 5a). The aggregation of AuNPs was readily seen by DLS (Figure 5b, Figure S23 in the Supporting Information), TEM (Figure 5c, also see Figures S24
S26 in the Supporting Information), and gel electrophoresis (Figure 5d), revealing a parallel-stranded poly(GA) homoduplex. Our work is significant and advantageous as compared to existing techniques in three major aspects: (1) It provides an unprecedented opportunity to place a conformational control on a nucleic acid duplex despite its thermodynamic preference, which should be adaptable for other similar structures. It is noteworthy that the capability of stabilizing parallel-stranded DNA or RNA duplexes is long expected5 for the investigations of 5070

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Figure 5. A gold nanoparticle based assay for the parallel-stranded conformation of a [(GA)12G]2 homoduplex. (a) Schematic drawings showing the role of AuNPs in this process. (b
d) DLS (see also Figure S23 in the Supporting Information), TEM (see also Figures S24
26 in the Supporting Information for TEM images of all related samples), and gel electrophoresis evidence. Figure S22 in the Supporting Information showed the formation of a [(GA)12G]2 homoduplex by a nondenaturing PAGE.

their possible biological roles and potential biomedical implications. (2) It provides a very straightforward way to probe DNA strands’ orientations, while other spectroscopic techniques usually need significant preknowledge about the spectral signatures of the system under investigation in order to make a correct judgment or suffer from interference from coexisting substances. We should mention that the spectroscopy based methods such as CD are sometimes superior in that no DNA labelings are required, which might be beneficial for in vivo monitoring of DNA conformation change. (3) Our process can be coupled to various instrument-based or instrument-free detections, which is of great significance toward enhanced adaptability as well as lowered experimental costs. The signal readout routes of this method can be as simple and convenient as naked-eye based colorimetric, precipitation, and gel electrophoretic assays or as quantitative and accurate as UV
vis absorbance spectroscopy and a dynamic light scattering (DLS) measurement.

’ CONCLUSIONS In conclusion, we have developed a sensitive, simple, and extremely straightforward method that can be employed to distinguish and control the interstrand orientation of a DNA duplex. This method takes advantage of the often overlooked (or viewed as a negative effect in many situations) electrostatic and steric repulsive forces between highly charged AuNPs to realize such a determination. The strategy reported in this work should be similarly applicable to other DNA conformations (e.g., triplexes, quadruplexes, i-motif) and may be generalized to explore the structures of peptides and other natural/synthetic polymeric or supramolecular structures. We want to emphasize here that existing spectroscopy based benchmark methods for research on DNA conformations such as circular dichroism (CD) and F€orster resonance energy transfer (FRET) all belong to the class of passive techniques that can not be employed in an imaginably convenient way to directly control the formation of a

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DNA duplex with desired interstrand orientation. With the further employment of DNA monofunctionalized AuNPs,21
26,38,42,43 it is possible to isolate the hybridized dimeric assemblies23 followed by a competitive displacement of the DNA duplexes from the AuNPs by small thiol molecules or a chemical etching of the gold cores. In such a way, metastable DNA duplexes in unconjugated forms may be obtained for further structural and functional investigations. It is noteworthy that the use of monofunctionalized DNA
gold conjugates also removes a possible ambiguity in structural determinations when a higher order structure such as a four-strand G4-DNA exists, which would widen the applicability of our method. Furthermore, AuNPs should also be able to facilitate delivering an unusual DNA conformation into an in vivo environment to explore its biological effects.29
33 Therefore, we expect the gold nanoparticle based assay will be serving well as an ideal counterpart to existing methods toward conformational analysis, structural control, and functional explorations of chemically or biologically interesting DNA structures, which will enrich the scientific tools for chemical/molecular biology, biophysics, and biomedical research. Since the biological roles of some noncanonical DNA conformations and structures including parallel-stranded DNA are still not well understood, the analytical tool we have developed could be employed to build a highthroughput visual screening platform for the search of biologically important DNA sequences that may tend to adopt parallel stranded structures.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional experimental details, more TEM images, and DLS, PAGE, and optical absorbance data. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by NSFC (Grant Numbers 20873134, 91023005, and 20605019), the Fundamental Research Funds for the Central Universities (Grant WK2060190007), a CAS Bairen start-up, and PCSIRT (Grant IRT0756). The authors greatly appreciate D. Liu, Y. Yang, L. Gan, and Y. Tao for their expertise suggestions and help on the CD measurements. Prof. D. Liu and Prof. Y. Yang are greatly acknowledged for providing us access to their CD spectrometers. ’ REFERENCES (1) Shchyolkina, A. K.; Lysov, Y. P.; Il’ichova, I. A.; Chernyi, A. A.; Golova, Y. B.; Chernov, B. K.; Gottikh, B. P.; Florentiev, V. L. FEBS Lett. 1989, 244, 39–42. (2) Cubero, E.; Luque, F. J.; Orozco, M. J. Am. Chem. Soc. 2001, 123, 12018–12025. (3) Robinson, H.; Wang, A. H. J. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5224–5228. (4) van de Sande, J. H.; Ramsing, N. B.; Germann, M. W.; Elhorst, W.; Kalisch, B. W.; von Kitzing, E.; Pon, R. T.; Clegg, R. C.; Jovin, T. M. Science 1988, 241, 551–557. (5) Ramsing, N. B.; Jovin, T. M. Nucleic Acids Res. 1988, 16, 6659–6676. 5071

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