1.4. Artificial Base-Pairs Based on Hydrogen Bonding Interaction
1.4.3. Size Expanded Base Pairs
polymerase.26a-b Pyrrolo-dC has excitation and emission maxima at 350 nm and 460 nm, respectively, which, like the previously described analogues, allows selective excitation in the presence of native nucleic acid bases and proteins. This base analogue can pair with guanine and like 2-AP, pteridine and hydrocarbon base analogues, shows significant quenching of fluorescence when incorporated into single and double stranded DNA. The quenching can be used to monitor local melting of the G:C base pairs in a DNA helix and can serve as a complementary probe to 2-AP, which reports on melting of AT base pairs.26c
Xanthosine Analogue: The base analogue of the rare base xanthosine (5-aza-7- deazaxanthine) (1.38) has been synthesized and reported by Benner et al. (Figure 1.11).27 This base was designed based on the supposition that the rare tRNA constituent wyosine carries the 5-aza-7-deazapurine substructure, and it is this structure that makes the base fluorescent. When excited at 250 nm, this base analogue displays two emission maxima at 410 nm and 580 nm.
O HO
HO N
N N
N H H
H H
O OH
OH N
X N
N O
O H
H
O OH HO N N
N O
O H
H
X = H, OH
1.42 (xanthosine analogue) 5-aza-7-deazapurine 2'-deoxyriboside 1.40 1.41
2'-deoxyxanthosine (X =N)
Figure 1.11: Structure and hydrogen bonding property of xanthosine analogue.
invented during that time. Leonard predicted that the stretched out adenine base would destabilize a natural DNA as it is too large as compared to natural DNA bases.
The synthesis and study of expanded DNA bases was done with the motive of expansion of genetic alphabets which is orthogonal to the natural one, to develop bases with greater electronic conjugation which can endow the nucleosides with fluorescence properties, to probe the steric effects of the active sites of polymerase enzymes and finally to develop a new helix that is thermodynamically more stable than the natural DNA due to the stacking interactions between the bases.29
Benzo Fused Molecular Designs
:
With the aim of generating size expanded DNA molecules the expanded analogues of the purines and pyrimidines containing the fused benzene ring have been synthesized by Kool et al. and their properties studied. The benzo fused analogues of purines and pyrimidines paired with natural DNA bases could potentially yield a regular helix with expanded diameter (Figure 1.11).30 The fusion of the benzo ring increases the size of the natural nucleoside by 2.4 Ǻ, with similar vectors of extension. Modeling studies as well as experimental studies have shown that the DNA backbone with benzo-fused nucleosides required only small adjustments of bond angles with no large change to sugar conformations in order to adjust the expanded nucleosides inside the helix duplex. However, the expanded DNA differs from the natural B- DNA in one respect: that it generates greater number of base pairs per turn creating larger outer circumference than that of the natural DNA.Modeling studies have suggested that there are 14 base pairs per turn as compared to the 10.5 base pairs in natural B-DNA.
While natural DNA is composed of four components with two types of ring systems, the purine and the pyrimidine rings, there are eight components of size expanded DNA, known as x-DNA with four types of ring systems. In x-DNA, the benzopurines are paired against pyrimidines and the benzopyrimidines are paired against purines. While the pairing selectivity in natural DNA comes from the complementary hydrogen bonding but in x-DNA, complementary hydrogen bonding as well as complementary size are the determining factors of pairing selectivity (Figure 1.12).30 c
N N O HO HO
N N N H H
N N O Me
O
H N
N O HO HO
N
N N
N O N
N H
H H O
H H
O HO HO
Me
HN N O
O H
N N N
N H N
H
O HO HO
HN N
O N
N N
N H N H
O H
N H
H dxA-T dxG-C dxA T dxG C
dxT-A dxC-G A dxC G
1.43 1.44
1.45 1.46
O OH
OH O
OH
OH
O OH
OH O
OH
OH
dxT
Figure 1.12: Structures and H-bonding pattern of x-DNA bases.
The x-DNA involved a linear extension of purine and pyrimidine by the addition of a benzene ring to the natural bases, a similar benzo-homologation with a different extension vector yielded the y-DNA which Kool et al. named as “wide DNA” (Figure 1.13).31 The extended conjugation of the expanded DNA bases due to the added benzo fusion rendered it fluorescent with a large Stokes shift of 50-80 nm in contrast to the naturally occurring bases.They are efficient fluorophores and fluoresce in the visible wavelength region with quantum yields between 0.30 and 0.6.31 a, c
O OH
OH O N
HO HO
N N O Me
O
H O N
HO
HO N
N N H
O OH
HO N
N N
N N H
O OH HO
N N H dyA-T dyG-C dyA T dyG C
dyT-A dyC-G
dyT A dyC G N
N N H H
N N N O
H H
H O H
N HN
O
O H
H
N HN
O
N H
H O
N H
H
N N
1.47 1.48
1.49 1.50
O OH
OH
O OH
OH
O OH
OH
Figure 1.13: Structures and H-bonding pattern of y-DNA bases.
The designed expanded bases that are singly substituted into natural DNA have been found to destabilize the DNA helix as the benzo-expanded base pair is too large which induces strain leading to backbone distortions. For the xDNA analogues, the destabilization penalty ranged from 0.3 to 1.7 kcal/mol relative to the stability of natural base pairs and for yDNA pairs, the range has been found to be 0.6-2.2 kcal/mol. However, replacement of natural bases with all unnatural size expanded base pairs the x-DNA and y-DNA have been found to form highly stable, sequence selective double helices. It is reported that the size-expanded bases form the components of artificial genetic system with eight components as compared to the four components in the natural genetic system.32 The high binding selectivity, affinity in base pairing and fluorescence property of expanded DNA bases might be useful in detection of nucleic acid sequences.
Naptho Fused Molecular Designs: Latter on, Kool et al. has developed more widened DNA by synthesizing expanded DNA base pairs via naptho-homologation.
Two naptho-homologated deoxyribonucleosides dyyT and dyyC which are expanded analogues of thymidine and cytosine thus have been synthesized and incorporated into oligonucleotides (Figure 1.14)33 and found that the inclusion of two benzene rings widened the dyyT and dyyC by about 4.8 Ǻ as compared to their natural counterparts.
Such type of DNA was named as “double wide DNA” by Kool et al. The extra conjugation rendered by the additional benzene ring makes the DNA bases fluorescent with red-shifted absorption and emission property.33 The fluorescence of yy-DNA bases suggests that such type of widened bases might find applications in detection and imaging of natural nucleic acids. However, in contrast to the standard nucleic acid probes, the yy DNAs do not give simple denaturation behaviour. The complex melting behavior indicates that the strong single-stranded stacking of the component bases might prevent the measurement of the affinity of yyDNAs for their complements using standard thermal denaturation methods.
O OH
O OH HO HO
HN N H O
O N
N N
N 4.8 Ao N
O HO HO
HN N O 4.8 Ao N H
H
N N
O
N N
N H yyT-A yyC-G
yyT A yyC G
1.51 1.52
O OH
OH
H H H
H
Figure 1.14: Structures and H-bonding pattern of doublewide DNA.