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NMR Spectroscopy and
Other Spectroscopic
Characteristics

4
...
3

NMR Spectroscopy

The precise three-dimensional (3D) conformation of nucleic acids at the atomic level is
investigated by nuclear magnetic resonance (NMR) spectroscopy
...
As the resonance frequencies are shifted in different chemical
environments, it is very useful to identify the chemical interactions around the target
atomic nuclei
...
As for the analysis, protons on base moiety from aromatic, amino, and imino
should be assigned (Figure 4
...
Compared with Watson–Crick base pairs forming a
canonical duplex, Hoogsteen base pair shows different chemical shifts of proton
especially for its imino group [6]
...
0 ppm) compared
with other protons in nucleic acids and different from the imino protons of Watson–
Crick base pairs (∼12
...
5 ppm) (Figure 4
...
Thus, the imino protons are used
as an index for the analysis of non-canonical structures
...
G4 represents the identical 4n (n:
the number of G-quartet) 1H NMR peaks in the region of the imino proton of Hoogsteen
base pairs
...
The imino protons between 15 and 16 ppm are characteristic
of an i-motif structure [8]
...
NMR signals can distinguish
these topologies from the difference of chemical shifts of imino protons
...
The mixture of the structures rules out the 4n peaks in the region of
imino protons
...

Moreover, the width of NMR peaks represents the relaxation of spin, indicating that the
slow replacement of proton from the solution makes a broad width of the peak
...
For further structural
analyses, 2D NMR analysis such as heteronuclear multiple quantum correlation
(HMQC) (information of direct bonding between carbon and hydrogen atom),
correlation spectroscopy (COSY) (information of geometry of hydrogen atoms), total
correlation spectroscopy (TOCSY) (information of geometry of hydrogen atoms in the
region of hydrogen-replaced carbon), and nuclear Overhauser effect spectroscopy
(NOESY) (information of the distance between hydrogen atoms) is performed to
determine the 3D structure of the non-canonical nucleic acids as well as the canonical
structure
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5 – 8
...
7 – 8
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0 – 1
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7 – 7
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6

O
CH3

N
N

O

13 – 14
NH

N

H

O

6
...
9
Sugar

6
...
9

Sugar

Duplex
(Watson–Crick bp)

G-quadruplex

i-Motif

15
(b)

13
1H

11

(ppm)

Figure 4
...
(a) NMR chemical shifts of the key
residues observed in 1 H NMR spectra of nucleic acid bases and sugar
...


depends on the number of hydrogen atoms
...


4
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4

Other Spectroscopic Characteristics of Non-canonical Nucleic

Acids
As Watson and Crick deduced the double helix structure of DNA, X-ray diffraction
methods provide concrete and precise information about the 3D structure of nucleic
acid structures (Figure 4
...
The diffraction method can solve the 3D structure at

Single-crystal X-ray diffraction
Crystal
X-ray

(a)
Small-angle X-ray scattering

Solution

X-ray

(b)

Figure 4
...

(a) Image of single-crystal X-ray diffraction
...


the atomic level by analyzing diffraction patterns of fiber and single crystal from
nucleic acids according to Bragg’s law n𝜆 = 2d sin 𝜃,where d is the spacing between
scattering planes (path difference), 𝜃 is the incident angle, 𝜆 is the wavelength of the
X-ray, and n is an integer
...
Therefore, the analysis for
non-canonical nucleic acids by X-ray is simple
...
Because the molecular environment of
the crystal is highly packed, it is possible that the crystal structure does not reflect the
true image of nucleic acids in solution
...
It is difficult to determine which structure is the major in the
physiological condition because the cellular condition is highly dense with
macromolecules (see Section 4
...
Thus, it is important to consider the 3D structure
from the viewpoint of the condition around target nucleic acids
...
5b)
...

Therefore, it is utilized to reveal conformational dynamics of non-canonical nucleic
acids relatively having a large molecular weight such as riboswitch and ribozyme,
which have difficulty in crystallization
...
Transmission electron
microscopy under a cryogenic condition (cryo-EM) is used to visualize the structure of
nucleic acids (Figure 4
...
Atomic force microscopy (AFM) also provides structural
information
...


Figure 4
...


3D structure
2D projection

Single-particle
analysis



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