The Base Pair Contents and Sequences of DNA Double Helixes

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

The Base Pair Contents and Sequences of DNA Double Helixes Differentiated by Surface-Enhanced Raman Spectroscopy Yang Li, Tianyang Gao, Guantong Xu, Xiaoxuan Xiang, Xiao Xia Han, Bing Zhao, and Xinhua Guo J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.9b00936 • Publication Date (Web): 15 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

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The Journal of Physical Chemistry Letters

The Base Pair Contents and Sequences of DNA Double Helixes Differentiated by Surface-Enhanced Raman Spectroscopy Yang Li, [a] Tianyang Gao, [a] Guantong Xu, [a] Xiaoxuan Xiang, [a] Xiaoxia Han, [a] Bing Zhao [a] and Xinhua Guo*[a], [b] a

State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin

University, 2699 Qianjin Street, Changchun 130012, P. R. China. b

Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education,

College of Life Science, Jilin University, Changchun 130012, P.R. China Corresponding Author * Dr. Xinhua Guo. Tel: 86-431-89228949; Fax: 86-431-89228949; E-mail: [email protected]

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ABSTRACT

Direct, label-free sequence analysis of DNA hybridization has been achieved by surface-enhanced Raman spectroscopy (SERS). In this work, aluminum-ions-aggregated and iodide-modified silver nanoparticles (Ag IANPs) were used as substrates for obtaining Raman spectra of the DNA strands with the same base composition but different sequences, which form random coils or various hairpin conformations. Upon DNA hybridization, reproducibly enhanced bands were easily observed, corresponding well to the formation of Watson-Crick hydrogen bonds, base ring-breath vibrations and hairpin loop. These characteristic bands can be used for unambiguously distinguishing the hairpins from the random DNA conformation. Moreover, by using the deoxyribose band (959 cm-1) as an internal standard to normalize the characteristic bands at 1703 cm-1 corresponding to dG νC=O H-bond, the Guanine-Cytosine base pairs contents and sequence in DNA hairpins can be accurately measured. Applying this method, a single base mutation in a functional double helix was confidently identified.

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As it directly relates to the diagnostic of many human diseases,1-3 detection of single-nucleotide polymorphisms (SNPs), DNA hybridization and instability is becoming increasingly important. Compared with traditional techniques such as polymerase chain reaction and microarray techniques, methods based on surface-enhanced Raman spectroscopy (SERS), providing a simple, rapid and low-cost way for DNA detections, are free of the use of fluorescent tags and able to give intrinsic chemical structure information of DNA molecule and DNA complex mixtures. Under this circumstance, various SERS strategies have been developed. 4-6 Although SERS signals produced by the interactions of an external molecule with the DNA molecule has been used to monitor the structural changes of the DNA molecule, the SERS signals produced by DNA molecule itself are more direct and highly-related to the intrinsic structure. Silver nanoparticles have been widely used for obtaining directly-enhanced DNA signals. 7-8 As the backbone phosphodiester groups of DNA molecules carry negative charges, the DNA molecule is rather difficult to be attached to the rough surface of the negatively charged nanoparticles. Therefore, Halas et al. used silver nanoparticle to analyze thiol-modified DNA molecules, they observed enhanced Raman signals of single-stranded DNA (ssDNA) and doublestranded (dsDNA).9 In this case, dominated bands were produced from the adenine (A) base. They further replaced all A bases in the complementary strand with an adenine base analogues (2aminopurine), and compared the changes of peak intensity of A base to detect DNA hybirilization.10 Following, Bell et al. introduced magnesium sulfate into the positively or the negatively charged silver sols, where the positively charged Mg2+ ions changed the negative surface of the sols, and the negatively charged sulfate altered the environment of the positive ones. As a result, direct SERS signals of ssDNA and dsDNA were achieved under this method, and a single-base-sensitivity as well as label-free detection of DNA molecules was realized.11-12


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Furthermore, Ren et al. introduced iodide ions as a cleaning agent for the surface of the silver nanoparticle, which effectively improved the reproducibility of the SERS signal of DNA. 13 They also used the SERS band of the phosphodiester group as an internal standard to normalized SERS signals of ssDNA and quantitatively analyze the base content in the same length of ssDNA. Recently, we reported an aluminum-ion-aggregated silver nanoparticle (Ag IANPs) by introducing aluminum ions into Ag@I (silver nanoparticles modified by iodide ion).14-15 With Ag IANP as substrates, we observed characteristic bands corresponding to the hydrogen bonds produced by cyclic guanine bases, the bands corresponding to glycosidic bond angles, the base ring of dG in DNA G-quadruplex conformation, and the featured band corresponding to C+ •C base pair in the i-motif structure. Thus, the Ag IANPs showed great potential of application for obtaining more structural information of DNA complexes. By comparing the band shift and intensities of adenine base signals appearing in the SERS spectra of single- and double-stranded DNA, Guerrini et al. detected the DNA hybridization and the base pair mismatching.16 Ren et al. used the ratio of the normalized relative intensity of A base to (A +C) bases to quantitatively detect the amounts of A or C base in the same length sequences.13 Furthermore, they subtracted each complementary sing-stranded spectrum from the doublestranded spectrum to achieve the DNA hybridization detection. To our best knowledge, so far, there is no report of using enhanced characteristic bands of double-stranded DNA for direct detection of DNA hybridization. In our previous work, by using Ag IANPs as substrates, the exclusive SERS bands corresponding to chemical information of the particular secondary DNA structures have been observed. We conceived that this nanoparticle could be further used to obtain the featured band of DNA double


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helixes. Hence, in this work, five sets of DNA strands were selected for our studies. Each set of DNA strands contains the DNA sequences with the same base composition but the different base sequence, which forms a DNA random coil or folds into a DNA hairpin (Table 1). By comparison of the featured bands produced from hairpin and random coil, we attempt to obtain DNA hybridization information. As expected, the characteristic SERS bands corresponding to DNA base pairs and hairpin loop have been clearly detected, which could unambiguously distinct the hairpin conformation from the random coil. Furthermore, we proposed to use the peak intensity of the deoxyribose at 959 cm-1 as an internal standard to optimize the SERS signals observed in the hairpin spectra, which allows for the precise determination of base-pair contents and sequence. Our approach was further extended to analyze dsDNAs, as a result, single base mutation was succesfully detected. Figure 1 shows the experimental procedure and the SERS spectrum of one set of DNA strands.


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Figure 1. (A) A schematic diagram of the experimental procedure for DNA SERS detection; (B) The SERS spectra of 1HP3 (DNA hairpin) (green) and 1L0 (ssDNA) (blue) normalized by the intensity of phosphate band at 1089 cm-1; Insert: The structural diagram of GC base pair. (C)The hot spots guided by aluminum ions on Ag@I. Ag@I: iodide-modified Ag nanoparticles. Ag@cit : silver citrate nanoparticles. First of all, two ssDNA, 1L0 and 1HP3 were selected for SERS detection (Table 1). These two strands have the same base composition but the different base sequence. In the same annealing and testing condition, 1L0 keeps a random coil conformation while the 1HP3 folds into a hairpin


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structure.17 The CD spectra of 1L0 and 1HP3 confirmed the formation of the hairpin conformation of 1HP3 by showing a positive band around 280 nm and a negative band around 245 nm,18 which could distinct it from the random coil formed by 1L0 (Figure S1A, Supporting Information). To our best knowledge, so far, no intensively characteristic SERS bands of the double helixes have been used to differentiate dsDNA from the ssDNA. Figure 1 shows the SERS spectra of 1L0 and 1HP3 normalized by the phosphate band at 1089 cm-1.13 Comparing two spectra, we can clearly see that the peak at 1703 cm-1, corresponding to the formation of the Watson-Crick hydrogen bond between guanine and cytosine bases, appears in the hairpin spectrum but does not show up in the spectrum of 1L0. At the same time, an enhanced T-loop peak at 620 cm-1 is also observed. These bands can serve as good markers for characterizing the formation of the DNA hairpin. In addition, we can see that after the hybridization, the peak at 654 cm-1 corresponding to guanine ringbreathing vibration is enhanced and shifts to 651 cm-1, which can be explained by the formation of triple hydrogen bonds between the guanine and cytosine. The peak intensity at 732 cm-1, corresponding to the adenine ring breathing vibration is also significantly enhanced, which is attributed to its participation in the formation of Watson−Crick hydrogen bond upon the folding of the DNA hairpin. According to the surface selection rules of SERS 19, the ordered A-T or G-C base pairs may stand on the surface of the substrate, resulting in the significantly enhanced ring breathing vibration peaks of the A and G bases. The peak at 959 cm-1 corresponding to the DNA deoxyribose backbone also undergoes this augmentation. These observations further support our views that Ag IANPs provides a better environment on its surface, which allows paired DNA bases to be located in the “hotspot”, largely enhancing Raman signals. 14-15 The featured SERS peaks of the DNA hairpin 1HP3 were assigned and listed in Table S1. To further confirm our observations, another set of DNA strands (2L0, 2HP4a and 2HP4b) with the same base composition was


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analyzed. These DNA sequences can form one linear structure and two different DNA hairpins, respectively. Their conformations were first confirmed by CD spectrum (Figure S1A). As expected, the same featured bands of 2HP4a and 2HP4b could differentiate the hairpins from the random coil 2L0 (Figure S2). However, by closely inspecting the SERS spectra of two hairpins 2HP4a and 2HP4b, we saw that two hairpins with the same amount of G-C and A-T base pairs gave enormous and unreasonable differences in intensity of G and A base ring breathing vibration peaks (Figure S2A). In contrast, in the bands normalized by the peak intensity of deoxyribose at 959 cm-1, the slight differences of intensities could be identified (Figure S2B). We realized that the characteristic peaks might be normalized in an inaccurate way and the deoxyribose peak (959 cm-1) could be more suitable as a normalization site than the phosphate group (1089 cm-1). Table 1. The name, sequences, chemical formula and possible GC base pair numbers of the DNA strands studied in this work. Name 5’-3’ Formula GC Pairs 1L0 CTCAGTCTTAGGTATT C3A3G3T7 no 1HP3 CTCAGATTTTTCTGAG C3A3G3T7 3 2L0 TCCAGTTGCGTATTCG C4A2G2T6 no 2HP4a GGACTGTTTTCAGTCC C4A2G2T6 4 2HP4b GCGAACTTTTGTTCGC C4A2G2T6 4 2HPA AAGGAATTTTTTCCTT C2A4G2T8 2 2HPB AAGAGATTTTTCTCTT C2A4G2T8 2 3HPA GGAGAATTTTTTCTCC C3A3G3T7 3 3HPB GAGAGATTTTTCTCTC C3A3G3T7 3 4HPA GGAAGGTTTTCCTTCC C4A2G2T6 4 4HPB GGAGAGTTTTCTCTCC C4A2G2T6 4 DS1 CTAGAGCTC C3A2G2T2 DC1 GAGCTCTAG C2A2G3T2 DX1 GAGCTATAG C2A2G3T2 DX2 GAGATCTAG C1A3G3T2 DX3 GAACTCTAG C2A3G2T2 DX4 AAGCTCTAG C2A3G2T2 DX5 GAGCTCTAA C2A3G2T2 DX6 GAGCTCTCG C3A1G3T2 -


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Figure 2. (A)The SERS spectra of the DNA hairpins formed by the six sequences 2HPA, 2HPB; 3HPA, 3HPB; 4HPA and 4HPB. The gray bars mark dG ring breathing, dA ring breathing, DNA deoxyribose and dG ν C=O (H-bond) from left to right. All peaks are normalized at the intensity of the peak at 959 cm-1. (B)The graph of relative intensity ratio (I1700/I959) versus the numbers of the GC base pair content (n) of the DNA hairpin formed by the six sequences. Each point represents the average of 5 measurements, and each error bar indicates the standard deviation. To verify the rationality of the normalization and to find the relationship between SERS bands and base pair sequence of hairpins, three additional pairs of hairpins 2HPA/2HPB, 3HPA/3HPB, and 4HPA/4HPB (Table 1) were further studied. Each pair of the hairpins has the same base composition, G-C and A-T base pair amount but different base pair order; different sets of haipins have different numbers of G-C pairs (DNA sequences are shown in Table 1 and the schematic diagram of the hairpin structures is shown in Figure S3). CD spectra (Figure S1B) confirmed the formations of the hairpin formations. Figure 2A shows SERS spectra of those hairpins (normalized at the intensity of deoxyribose band). This time, minor changes in the signals of each hybridized strands can be clearly identified. What’s interesting is that with the increment of the G-C pair content of each DNA strand from 2HPA to 4HPB, the peak intensity at 1703 cm-1 (ascribed to dG νC=O H-bond ) gradually increases as well. Figure 2B exhibits an incredible linear relationship – the error bar of each point is highly qualified and much more promising than the threshold required to discriminate the single base pair. This linearity indicates the possibility to establish a


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quantitative relationship between the relative intensity of the SERS peaks and different G-C base pair contents in the dsDNA strand. It is worth noting that we have also observed subtle but rather regular changes regarding to the ring breathing vibration intensity of A and G bases in the DNA hairpin structure with the same base composition. When bases with larger base stacking area are adjacent (the base stacking area: G>A>C>T

20),

the increscent base stacking area results in

increased SERS signal intensity of base ring peaks. Taking the 2HPA and 2HPB as an example, when the adjacent adenines (in 2HPA) are separated by guanine (in 2HPB), the ring-breathing vibration peak intensity of adenine is enhanced as the neighbor AA bases become GA bases, while that of the guanine decreases as the neighbor GG bases became GA bases. This rule obtained from the SERS spectra of the DNA hairpins with the same base pair number but the different base order allows us to detect the base pair sequence of DNA double helixes with the same base content, even the very minor structural difference. To demonstrate the feasibility of this method, we selected one DNA fragment as our DNA focus: DS1, a primer (CZ 447) in the PCR, comprises the cleavage site of SacI and XbaI, two endonucleases,21 and then we selected its complementary sequences DC1 and DX1 to form double helixes. The difference of DC1 and DX1 is that the sixth cytosine (from 5’ to 3’) in DC1 is replaced by adenine (Table 1), so that DX1 does not exactly match with DC1. Figure S4 shows that one certain pair of bases has muted into an adenine-guanine base pair in DS1-DX1, which is not able to form the hydrogen bond with each other. As it is illustrated in the SERS spectra of DS1-DC1 and DS1-DX1 (Figure 3), normalized at the peak intensity of the deoxyribose, the peak intensity of 1703 cm-1 sharply decreases due to the presence of the unpaired bases in the dsDNA, accurately indicating the change of G-C pair content. At the same time, the intensity of cytosine ring breathing peak decreases while that of adenine increases, consisting with the sequence change, where one


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cytosine base in DC1 is replaced by an adenine base. Furthermore, one can see that there is no change in the intensity of guanine ring breathing peak after a base mutation. Following the rule above, if the fourth (from 5’ to 3’) cytosine base has been replaced, the stacking area of G-A will increases compared to that of G-C, resulting in an enhancement of G-ring breathing peak. Therefore, we can reach the conclusion that it is the cytosine located at the sixth position (from 5’ to 3’) has been replaced by an adenine. Also, an increment of the peak intensity at 1347 cm-1 representing the thymine deoxyribonucleotide can be attributed to the effect of larger base stacking of T-A than that of T-C.

Figure 3. The SERS spectra of the dsDNAs DS1-DC1 and DS1-DX1 normalized at the peak intensity of deoxyribose at 959 cm-1. To further prove our hypothesis, five more dsDNA：DS1-DX2, DS1-DX3, DS1-DX4, DS1DX5 and DS1-DX6 were selected for studies. From 5’ to 3’, in each helix one original base of DC1 was replaced, which is the shifting of the fourth C to A for DX2, the change of the third G to A for DX3, the conversion of the first G to A for DX4, the change of the last G to A for DX5 and


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the change of the eighth A to C for DX6 (see Table 1 for sequence details). Figure 4A exhibits the spectra of DS1-DX2 and DS1-DC1, by comparing them one can see that the intensity of adenine ring breathing peak increases while that of the cytosine decreases in DS1-DX2, due to the mutation of C to A base in DX2 strand. At the same time, the intensity of guanine ring breathing peak slightly increases. This consists with that the larger stacking area of G-A than G-C. Also, the intensity of the peak at 1703 cm-1 (attributed to the G-C pairs) decreases sharply due to the loss of G-C content.


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Figure 4. The SERS spectra of dsDNAs (A) DS1-DC1 and DS1-DX2; (B) DS1-DC1 and DS1DX3; (C) DS1-DC1, DS1-DX4 and DS1-DX5; (D) DS1-DC1 and DS1-DX6. All the SERS spectra normalized at the peak intensity of deoxyribose at 959 cm-1. Figure 4B shows the compared spectra of DS1-DC1 and DS1-DX3. Due to the mutation of guanine to adenine, the intensity of ring breathing peak of adenine increases while that of the guanine gets down; also, that of the cytosine decreases as well, which results from the less stacking area of A-C compared to G-C. What’s more, the intensity of peak at 1703 cm-1 gets lower because


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of the decline of G-C content. However, the picture in DS1-DX6 is quite opposite (Figure 4C): after the mutation of adenine to cytosine, as the G-C content remains the same, the peak intensity at 1703 cm-1 undergoes nearly no difference. We can also tell the drop of the intensity of ring breathing peak of adenine, and the minor increment of that of the cytosine. Since the peak at 789 cm-1 reflects the presence of thymine as well as cytosine, this may be attributed to the fact that the peak intensity of ring breathing of neighboring thymine decreases. Also, for the stacking area reason, the peak for guanine has a certain decline. To have a deeper insight to the base mutation occurred at 5’ and 3’ end of the DNA strand, the DS1-DX4 and DS1-DX5 were analyzed. As shown in Figure 4D, besides the increment of the ring breathing peak of adenine and guanine caused by the mutation, we also find that the peak of adenine in DS1-DX4 is more intensive than that in DS1-DX5. This phenomenon may be due to the structural difference where the adenine is located at 5’ or 3’ end, since the mutated adenine bases possess the identical environment of adjacent bases. Again, because of the loss of G-C pairs in both strands, the peak intensity at 1703 cm-1 undergoes a certain decline. It is also worth noting that the peak at 1703 cm-1 in DS1-DX5 is slightly higher than that in DS1-DX4, which may be attributed to the presence of the G-C pairs at 3’ end. Based on the discussion above, we are confident to say that our method is capable of the precise and sensitive determination of base mismatch in the short dsDNA with the single-base sensitivity. It can not only tell its presence in the dsDNA, but also determine its specific position in the strands with no extra efforts, which is of great significance in bioscience analysis and forensic science. In summary, with the aluminum ions working as aggregating agents in the SERS analysis, we have successfully developed the method applicable for reliable, sensitive and truly label-free determination of double-stranded DNA hybridization in aqueous solutions. Our method not only


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has been proved efficacious in the precise determination of G-C content in DNA hairpin but also reveals the rules with regard to the base stacking by normalizing the SERS peak intensity at 959 cm-1 representing the deoxyribose of nucleotides. We further apply this method to get a clear observation of the presence of base mismatches in double helix DNAs and precise identification about the position of the wrong-paired bases. Our method, with more reliable and accurate results as well as much easier experiment process, will be of great value and implication in the biochemical and genetic analysis. Also, the methodologies presented in this work will surely provide more perspectives in the study of the sequence readout and structure study of nucleic acids and enrich the feasibility of SERS in biomolecule analysis. ASSOCIATED CONTENT Supporting Information. Detailed description of the experimental method; CD spectral data; supplement of SERS spectrum; schematic diagram of DNA hairpin structure. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENT


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This work was financially supported by the National Natural Science Foundation of China (21675060 and 21874054) and the International Collaboration Project of Jilin Province (20160414010GH). REFERENCES (1) Brooks, T. A.; Kendrick, S.; Hurley, L. Making sense of G ‐ quadruplex and i ‐ motif functions in oncogene promoters. The FEBS journal 2010, 277, 3459-3469. (2) Brooks, T. A.; Hurley, L. H. The role of supercoiling in transcriptional control of MYC and its importance in molecular therapeutics. Nature Reviews Cancer 2009, 9, 849-861. (3) Hursting, S. D.; Nunez, N. P.; Patel, A. C.; Perkins, S. N.; Lubet, R. A.; Barrett, J. C. The utility of genetically altered mouse models for nutrition and cancer chemoprevention research. Mutat. Res., Fundam. Mol. Mech. Mutagen. 2005, 576, 80-92. (4) Kang, T.; Yoo, S. M.; Yoon, I.; Lee, S. Y.; Kim, B. Patterned multiplex pathogen DNA detection by Au particle-on-wire SERS sensor. Nano Lett. 2010, 10, 1189-1193. (5) Laing, S.; Gracie, K.; Faulds, K. Multiplex in vitro detection using SERS. Chem. Soc. Rev. 2017, 45, 1901-1918. (6) Lane, L. A.; Qian, X.; Nie, S. SERS Nanoparticles in Medicine: From Label-Free Detection to Spectroscopic Tagging. Chem. Rev. 2015, 115, 10489-10529. (7) Rusciano, G.; Luca, A. C. D.; Pesce, G.; Sasso, A.; Oliviero, G.; Amato, J.; Borbone, N.; D’Errico, S.; Piccialli, V.; Piccialli, G. Label-Free Probing of G-Quadruplex Formation by Surface-Enhanced Raman Scattering. Anal. Chem. 2011, 83, 6849-6855. (8) Yang, S.; Dai, X.; Stogin, B. B.; Wong, T. S. Ultrasensitive surface-enhanced Raman scattering detection in common fluids. Proc. Natl. Acad. Sci. U. S. A. 2015, 113, 268-273. (9) Aoune, B.; Dongmao, Z.; Felicia, T.; Halas, N. J. Surface-enhanced Raman spectroscopy of DNA. J. Am. Chem. Soc. 2008, 130, 5523-5529. (10)Aoune, B.; Halas, N. J. Label-free detection of DNA hybridization using surface enhanced Raman spectroscopy. J. Am. Chem. Soc. 2010, 132, 12792-12793. (11)Papadopoulou, E.; Bell, S. E. Label-free detection of nanomolar unmodified single- and double-stranded DNA by using surface-enhanced Raman spectroscopy on Ag and Au colloids. Chem. - Eur. J. 2012, 18, 5394-5400. (12)Papadopoulou, E.; Bell, S. E. Label‐free detection of single‐base mismatches in DNA by surface‐enhanced Raman spectroscopy. Angew. Chem., Int. Ed. 2011, 50, 9058-9061. (13)Xu, L. J.; Lei, Z. C.; Li, J.; Zong, C.; Yang, C. J.; Ren, B. Label-free surface-enhanced Raman spectroscopy detection of DNA with single-base sensitivity. J. Am. Chem. Soc. 2015, 137, 5149-5154. (14)Li, Y.; Han, X.; Yan, Y.; Cao, Y.; Xiang, X.; Wang, S.; Zhao, B.; Guo, X. Label-Free Detection of Tetramolecular i-Motifs by Surface-Enhanced Raman Spectroscopy. Anal. Chem. 2018, 90, 2996-3000. (15)Li, Y.; Han, X.; Zhou, S.; Yan, Y.; Xiang, X.; Zhao, B.; Guo, X. Structural Features of DNA G-Quadruplexes Revealed by Surface-Enhanced Raman Spectroscopy. J. Phys. Chem. Lett. 2018, 9, 3245-3252.


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The Base Pair Contents and Sequences of DNA Double Helixes

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