1.8. Applications of Unnatural Nucleobase Analogues
1.8.3. Applications in Studying the Photophysical Processes in Fluorescent Unnatural DNA
Fluorescence techniques, now a days, find widespread application in biochemical and biophysical studies of macromolecules, specially, in studies of nucleic acids, fluorescence spectroscopy provides an important tool for the detection and probing of structure, dynamics and interbiomolecular interactions. Highly fluorescent dyes or nucleotide base analogues or fluorescently labeled nucleobases can serve as extremely sensitive probes in nucleic acid systems and enable experiments to be carried out in solution. Therefore, the lack of naturally occurring fluorescent bases has spurred the development of new modified nucleosides over the years with bright fluorescence properties which will be the probe for DNA analysis and can vastly be used in biological chemistry, biotechnology, biophysics and in light harvesting DNA based materials. Now a days, fluorescence based gene detection schemes are used routinely in numerous applications, such as DNA sequencing,78 in situ genetic analysis, SNP typing through techniques like fluorescence in situ hybridization (FISH),79 and highthroughput screening (HTS)80. Therefore, a major fraction of laboratories in the chemical, biological, and biomedical sciences make use of fluorescence methods and of fluorophores as reporters for elucidating the structure, functions and dynamics of biomolecules and biomolecular interactions.
Despite the large amount of research efforts undertaken at making new classes of fluorescent labels, commonly available organic fluorophores still suffer from a number of limitations such as the fluorophores often possess widely varied absorption maxima.81 Therefore, to address the limitations of the available fluorophores for practical applications, a number of significant approaches including the development of quantum dots82 and FRET dye pairs83 have been undertaken by several research groups. Moreover, as a result of tremendous research efforts a large number of fluorescent nucleosides/nucleoside base surrogates as novel intrinsically fluorescent nucleosides/base surrogates have been designed and exploited for the applications in fluorescence based techniques. As for example, a number of donor-acceptor labeled fluorescent oligonucleotide probes have been designed to study the photophysical phenomena like Förster resonance energy transfer (FRET) process, exciplex emission etc. during the course of hybridization event. Kool et al. have designed DNA probe containing an assembly of ~3−5 fluorescent nucleosides decorated with aromatic hydrocarbons and heterocycles as nucleobases. The fluorophores are arranged in a DNA-like chain, which they called as an “oligodeoxyfluoroside” (ODF).63a,84 These fluorophores are found to engaged in efficient electronic communication of excitation energy leading to the occurrence of multiple forms of energy transfer, including FRET, exciplex, excimer, H-dimer, and other mechanisms via close interactions of such flat aromatic fluorophores.84b
Förster resonance energy transfer (FRET) is a process in which energy is transferred non-radiatively from an excited donor molecule to an unexcited acceptor molecule over distances longer than their collisional diameters. FRET being a distance dependent process is used as a ‘spectroscopic ruler’ for determining the distance between a donor and an acceptor leading to the investigation of structure/dynamics of DNA. Towards this end, several donor-acceptor labelled fluorescent DNA probes have been designed for DNA analysis via generation of FRET signal. As for an example, dual labeled DNA containing pyrene as donor and perylene as an acceptor chromophore has been utilized by Shimadzu et al.85 for investigating nucleic acid hybridization assay (Figure 1.36). Thus, in this system, FRET with 100% efficiency has been observed upon hybridization of a target 32-mer
and its complementary two 16-mer deoxyribonucleotides whose neighboring terminals are labeled with a pyrene and perylene residues, respectively. Upon excitation at the absorption maximum of donor chromophore pyrene (λex=347nm), FRET emission from perylene is observed at 459 nm.
O O O
N NH O
O
O O O O
N NH O
O P
S O H O N O
P O O
O C 3' AAACTAGGGCCGCG
CH2
2' or 3' attach ment
C (CH2)2 O O P O
O O
TCTAGGCTTGCGTTA 5'
A T T G C G T T C G G A T C T CT G C G C C G G TA C A A A
AT A C G C A A G C TC A AG A G C G C G G C TC A TG T
T TTT
FRET
hn1
hn2
5' 3'
3' 5'
Pyrene labelled probe Perylene labelled probe
Target nucleotide
5' 3' 1.136
1.137
Figure 1.36: FRET from pyrene (donor) to perylene (acceptor) in nucleic acid Polyfluorophores labelled DNA have been designed by Kool et al. to study the photophysical aspects of labelled DNA. In the design of polyflorous DNA, Kool et al.
have shown that oligomeric exciplex and excimer forming fluorophores conjugated to DNA have the potential to act as donors for Förster resonance energy transfer process (Figure 1.37).86 The Kool’s designed oligodeoxyfluorosides (ODFs) contained stacked, electronically interacting fluorophores replacing the nucleobases on a DNA scaffold. They have designed twenty tetrameric ODNs containing a combination of monomer chromophores which includes pyrene, benzopyrene, perylene, dimethylaminostilbene, and a nonfluorescent spacer. These chromophores are conjugated in varied combinations at the 3′-end of a 14-mer DNA probe sequence.
They observed characteristic excimer and exciplex emission in the absence of an
acceptor chromophore in many of their designed ODF-DNAs.86 They also have investigated the ability of the twenty ODFs to donate energy to Cy5 and TAMRA dyes conjugated to a complementary strand of a target DNA. The acceptor dyes have been placed either at the near or far end of the ODF-conjugated probes in their design to test the possibility of a FRET process. Thus they are the first to establish the fact that the excimer/exciplex bands can act as donor from where the energy is transferred to the acceptor chromophores Cy5 and/or TAMRA leading to an FRET emission from the acceptor at very long wavelength region (Figure 1.37). This phenomenon has been observed for few of their designed multichromophore labeled probe DNA (ODF). They have concluded that ODFs may be candidates of “universal FRET donors” which could allow multicolor FRET of multiple species using only one excitation.
N N
HO O
O O O
P O OO O
O OP O
O O
O P O O
O O
OP O O
O
FRET
1.139
1.138
Figure 1.37: Schematic illustration of FRET in oligodeoxyfluorisides ODFs.
Excimer is the complexation between the ground state and excited state of the same chromophore while exciplex is the comlexation between excited state of a donor/acceptor and the ground state of an acceptor/donor chromophore.The excimer and exciplex formation has rigorously been studied in context of DNA in order to
probe the inter-biomolecular interaction and in fluorescence based detection of biomolecules.
As for example, excimer fluorescence emission have been applied for the detection of insertion/deletion (indel) polymorphisms that occupy approximately 10%
of all the polymorphisms in the human genome87a-c, and lead to serious gene expression errors because they often cause a translational frame shift and create premature proteins. Saito et al., have designed a Py2Lys-containing (1.139, Figure 1.38) ODN, 5'-d(GTGTTAAGCCPy2LysGCCAATATGT)-3'.87d-e With excitation at 350 nm, the fluorescence of the single-stranded ODN has been found negligible.
When Py2Lys-containing ODN is hybridized with 5'-
d(ACATATTGGCGGCTTAACAC)-3', which does not possess the base opposite to
Py2Lys, the fluorescence still remains weak . In contrast, the fluorescence spectrum of the duplex with 5'-d(ACATATTGGCAGGCTTAACAC)-3', where A is the base opposite to Py2Lys exhibits a strong fluorescence peak at 495 nm that is corresponding to the fluorescence wavelength from a pyrene excimer (Figure 1.34). The clear change in the fluorescence that depends on the presence or absence of the inserted base opposite to Py2Lys (A bulge) is useful for the detection of insertion polymorphisms. They have tested the detection of an insertion mutation by hybridization of Py2Lys-containing probe using the coding sequence of the epithelial sodium channel b subunit (bENaC) gene associated with Liddle's syndrome, which is an autosomal dominant form of hypertension with variable clinical expression.
Py2Lys-containing probe, 5'-
d(CTCACTGGGGTAGGGCCCAGTPy2LysGTTGGGGCT)-3', has been prepared and hybridized with bENaC gene sequences, 5'-d(AGCCCCAAC(G)n
ACTGGGCCCTACCCCAGTGAG)-3' (wild type, n = 0; G-inserted mutant, n = 1). It has been observed that the fluorescence emission from the duplex with a G-inserted strand is very strong and clearly distinguishable from the poor fluorescence of the duplex with a wild type strand. The hybridization of Py2Lys-containing ODN with a target DNA facilitates the determination of the presence/absence of insertion polymorphisms located at a specific site on the target DNA by a simply mixing.87 d-e
The exciplex emission in modified nucleoside containing DNA has been exploited by Saito et al. for the detection of target DNA in their designed oligonucleotide probe
O P O O O
N O
O NH
O HN O
Py2Lys (n=3)
n
1.140 (a)
(b)
containing methoxy-substituted fluorescent guanosine derivarive, 8-MPEG (1.140, Figure 1.39) These methoxy-substituted fluorescent guanosine derivarives have an intriguing property, i.e., they possess lower oxidation potentials than native guanosine and emit strong fluorescence at more than 380 nm.
It has been observed that the fluorescence of single stranded ODN 1 (X = 8-MPEG) is relatively weak and appeared at around 380 nm. When the opposite base of a complementary strand is adenine (N = A) in a duplex ODN 1 (X = 8-MPEG, 1.140) /ODN 2 (N = A), a new and strong emission is observed at a longer wavelength around 440 nm (Figure 1.39). The emission at 435 nm is not observed when the opposite bases of complementary strands are G, C and T, suggesting that the new emission is an exciplex emission from a complex formed between 8-MPEG and adenine (A).88 The stacking interaction between electron donating guanosine 8-MPEG of the probe DNA and adenine base of target DNA is the responsible factor for the formation of an exciplex upon photoexcitation of 8-MPEG.
Figure 1.38: (a) Schematic representation of the structure of Py2Lys (1.139) and hybridization of Py2Lys-containing probe an ODN with and without A bulge. "
A more prominent example of exciplex formation is observed from methoxy- substituted naphthalene derivative 8-MNEG (1.141, Figure 1.39). Thus, a characteristic fluorescence from the naphthalene chromophore appeared at 390~430 nm for single stranded ODN 1 (X = 8-MNEG). However, in a duplex [ODN 1 (X = 8-MNEG) /ODN 2
NH N N
O
NH2 N O HO
OH
H3CO NH
N N
O
NH2 N O HO
OH H3CO
1.141 1.142
DNA sequences used:
ODN 1: 5'-CGCAAT X TAACGC- 3' (X =8-MPEG,8-MNEG ODN 2: 3'-GCGTTA N ATTGCG- 5' ( N = A, G, C, T )
(N = A)], a new exciplex emission at 470 nm emerges together with the appearance of original naphthalene fluorescence, suggesting a partial formation of excited state complex (Figure 1.38). The formation of ground state complex between the naphthalene chromophore and A is indicated by the appearance of a new absorption band in the UV-visible spectrum of the duplex ODN 1 (X = 8-MNEG) /ODN 2 (N = A).
The fluorescence color change is also observable by naked eye upon illumination with 365 nm transilluminator. The fluorescence lifetime of double stranded ODN 1 (X = 8-
MNEG) /ODN 2 (N = A) with a biexponential decay has been measured. The longer lifetime component (τ = 2.2 nsec) is ascribable to the formation of an exciplex between 8-MNEG and A, whereas the shorter lifetime component (τ = 1.1 nsec) resulted from the naphthalene chromophore of 8-MNEG. The study of fluorescence lifetime indicated the existence of two species that are responsible for the fluorescence emission and suggest that longer lifetime component with a strong emission at 470 nm is due to the formation of an exciplex between 8-MNEG and adenosine (A).88
Figure 1.39: Exciplex formation between adenine and guanine derivative.