Bridged
Nucleic Acids (BNAs) as Molecular Tools
Sung-Kun
Kim, Klaus D. Linse, Parker Retes, Patrick Castro, Miguel Castro
Sung-Kun
Kim, Parker Retes, Department of Natural Sciences, Northeastern State
University, 600 North Grand Avenue, Tahlequah, OK 74464, United States
Klaus
D. Linse, Patrick Castro, Miguel Castro, Bio-Synthesis Inc, 612 East Main
Street Lewisville, TX 75057, United States
Correspondence to: Sung-Kun Kim, Department of
Natural Sciences, Northeastern State University, 600 North Grand Avenue,
Tahlequah, OK 74464, United States
Email: kim03@nsuok.edu
Telephone: +1-972 420 8505
Fax: +1-972 420
0442
Received: June 3, 2015
Revised: July 28, 2015
Accepted: August 2, 2015
Published online: September 22, 2015
ABSTRACT
There has been a
growing interest in developing chemically modified nucleotides for diagnostics
or therapeutics. Among the list of the artificial nucleotides, bridged nucleic
acids (BNAs) appear to be the most promising new generation BNAs to date in
view of the usage for applications. Here we briefly introduce the modified
nucleotides and the applications of the new generation BNAs. For the possible
applications, BNAs can be used for antisense, antigene, aptamer development, and
real-time clamp technology. Eventually, BNAs may play a pivotal role in a new
type of disease diagnostics and therapeutics in the foreseeable future.
© 2015 ACT. All
rights reserved.
Key words: BNA; Antisense; Aptamer; Real-time PCR clamp; Nucleic
acid analogs
Kim SK, Linse KD,
Retes P, Castro P, Castro M. Bridged Nucleic Acids (BNAs) as Molecular Tools. Journal of Biochemistry and Molecular Biology Research 2015; 1(3): 67-71
Available from: URL: http://www.ghrnet.org/index.php/jbmbr/article/view/1235
INTRODUCTION
With the completion of the human genome sequence, attention has been
paid to the utilization of this sequencing information to genetic medicines,
tailor-made medications and molecular targeted therapies. This has led to
encourage scientists to develop artificial nucleic acid analogs. The artificial
nucleic acid analogs potentially have many applications in molecular biology
and genetic diagnostics and have emerged in the field of molecular medicine.
The key importance of the artificial nucleic acid analogues is that when the
modified oligonucleotides form DNA:DNA and DNA:RNA duplexes, the binding
affinity is more stable than that of unmodified oligonucleotides[1-5].
Due to such an advantage, a variety of nucleic acid analogs have been synthesized
to enhance high-affinity recognition of DNA and RNA targets and to increase
duplex stability and increasing cellular uptake. However, although a large
number of chemically modified oligonucleotides have been developed during the
last few decades, most of these molecules have failed to give the desired
response excluding a few molecules. Among the list of the initially successful
nucleic acid analogs were peptide nucleic acids (PNAs),
2’-fluoro-3’-aminonucleic acids, 1’, 5’-anhydrohexitol nucleic acids (HNAs),
and locked nucleic acids (LNAs) (Figure 1). Peptide nucleic acids (PNAs) are
synthetic polymers that contain a peptide backbone and nucleic acid bases as
side chains[6,7]. These peptide based nucleic acid mimetic polymers
can form strong specific hydrogen bonds with complementary sequences present in
double-stranded DNA (dsDNA)[8]. 2’-fluoro-3’-aminonucleic acids are
modified nucleotide analogs that contain fluorine at the 2’ position[9].
HNAs are oligonucleotides built up from natural nucleobases and a
phosphorylated 1,5-anhydrohexitol backbone, where 1’,5’-anhydrohexitol
oligonucleotides can be synthesized using phosphoramidite chemistry and
standard protecting groups[10-14]. One dramatic improvement was made
with the introduction of the bridged nucleic acid, 2’, 4’-BNA, also called LNA[15].
The compound shows a better hybridization affinity for complementary strands,
both for RNA and DNA strands, in comparison with unmodified nucleotides.
Furthermore LNA can be used to design sequence selective LNA-oligonucleotide
hybrids that are soluble in aqueous solutions and exhibit improved biostability
compared with natural nucleotides[16]. The LNA monomer has now been
widely used in nucleic-acid-based technologies. However, there is a need for
further development because the nuclease resistance of the LNA is significantly
lower than that obtained by phosphorothioate oligonucleotide and the fact that
oligonucleotides containing consecutive LNA units or a fully modified
oligonucleotide using the analog are very rigid and inflexible[17].
Furthermore, additional research has proven that at least one kind of
LNA-modified antisense oligonucleotide is hepatotoxic[18]. After
developing the aforementioned modified nucleic acid analogs, other chemically
modified nucleic acids have been designed; for example, 2’-O,4’-C-ethylene
bridged nucleic acid (ENA)[19], and unlocked nucleic acid (UNA)[20]
have been developed (Figure 1). In the case of ENA, Morita et al showed
that the fact that the 2’-O,4’-C-ethylene linkage restricts the sugar puckering
to the N-conformation generates a high binding affinity for a complementary RNA
strand (i.e., Tm value increases by approximately 5℃ per modified
base) and enhances the resistance to nuclease digestion substantially[19].
Meanwhile, UNA is highly flexible due to the lack of C2’-C4’ bond present in
beta-D-ribofuranose as shown in Figure 1. UNA monomers have proven useful for
siRNA in combination with LNA, but rigorous cytotoxicity studies have not been
performed[21].
The currently
most promising nucleic acid analogs, bridged nucleic acids (BNAs), are
molecules that can contain a five-membered or six-membered bridged structure[15,22-26];
the comparison of BNA with other nucleic acid analogs is listed in Table 1.
After the birth of the 2’,4’-BNA (also known as LNA)[15], the
structure has evolved through the modification of the linkage connected at the
2’,4’-position of the ribose ring. As a result, 3’-amino-2’,4’-BNA and 2’,4’-BNACOC
were born as the 2nd generation of BNA[24,25]. To date,
the most updated version of BNA, the 3rd generation of BNA, is 2’,4’-BNANC
(2’-O,4’-C-aminomethylene bridged nucleic acid), containing a six-membered
bridged structure with an N-O linkage[27]. Specifically, the new
generation 2’,4’-BNANC includes 2’,4’-BNANC[NH],
2’,4’-BNANC[NMe], and 2’,4’-BNANC[NBn][27];
the structures of the BNA analogs are shown in figure 2. As for the
characteristics of 2’,4’-BNANC, when the BNAs are added to DNA or
RNA oligonucleotides, the degree of morphological freedom of the
oligonucleotides is restricted by its six member methylene bridge structure.
Thus, 2’,4’-BNANC bases are stacked in the A type conformation by
Watson-Crick hybridization, improving hybridization specificity and duplex
stability[27]. Moreover, the addition of 2’,4’-BNANC
enhances the Tm value by 2-9 ℃ per base[27]. The
applications using BNANC are discussed as below.
Antisense
and antigene applications
Oligonucleotides have been used to govern gene expression as
therapeutic tools for biological studies[28-30]. Antisense and
antigene technologies are excellent examples for therapeutic application, where
the combination of natural and modified oligonucleotides that has the ability
to form RNA duplexes or triplexes with a great affinity and stability. In
antisense technology, the formation of oligonucleotide RNA would block the
translational process or cleave the target mRNA with small interfering RNA (siRNA);
hence, the level of mRNA expression can decrease significantly. Meanwhile, in
antigene technology, a formation occurs between a single stranded
homopyrimidine triplex-forming oligonucleotide and a homopurine-pyrimidine
stretch in target DNA through Hoogsteen hydrogen bonding, where T•A:T and C•G:C base triplets
are formed[31]. Thus, it is not inconceivable that the triplex
formation can inhibit the binding of RNA polymerases in the transcription
process, leading to downregulation of target gene expression to a certain
extent. To accomplish this purpose, it is imperative that the chemically
modified nucleotides bind to their target mRNA or duplex DNA with a great
affinity.
Among the
chemically modified nucleotides, 2’,4’-BNANC nucleotides display
improved duplex and triplex affinity due to the reduction of repulsion between
the negatively charged backbone phosphodiester groups. Owing to the structural
advantage, the hybridization affinity to target RNA molecules has
unprecedentedly improved[32]. Several lines of evidence indicate
that the Tm value of 2’,4’-BNANC bases against complementary RNA
enhances +2 to +10 ℃ per 2’,4’-BNANC monomer[19,27,33,34].
In addition to the high affinity, incorporation of 2’,4’-BNANC bases
into oligonucleotides results in the increased resistance to endo- and
exonucleases[27]. As another example for antisense therapeutics
using BNA, BNA-based antisense successfully suppresses hepatic PCSK9 expression
that leads to a significant reduction of the serum LDL-C levels of mice, where
these findings support the notion that BNA-based antisense oligonucleotides can
induce a cholesterol-lowering action in hypercholesterolemic mice[35].
Aptamer
capping applications
Aptamers, oligonucleotides binding to their target
molecules with high affinity, are used as research materials in either
diagnostic or therapeutic applications. The aptamer oligonucleotides, a library
of single-stranded oligonucleotides, usually contain 30 to 60 bases in length
using Systematic Evolution of Ligands by EXponential enrichment (SELEX)
technique, where the technology initially begins with a random pool of
oligonucleotides and finally ends with tightly binding oligonucleotides[36,37].
A caveat is that the resulting oligonucleotides are susceptible to nucleases so
that the oligonucleotides readily degrade. In order to address this issue, the
capping of the ends of aptamers can be used as a solution. The number of
enzymes that are able to incorporate modified nucleotides were very limited;
however, the process of capping 2’,4’-BNANC monomer can be made by a
one step enzymatic reaction using 2’,4’-bridged nucleoside 5’-tripohsphate and
terminal deoxynucleotidyl transferase[38]. As an example, Kasahara et
al. reported that the capping of the 3’-end of the thrombin aptamer with
2’,4’-BNANC bases significantly enhances the nuclease resistances
and binding affinity in human serum[39]. In addition to these
improvements, the binding ability of the aptamers was not influenced by the
capping of 2’,4’-BNANC bases[39].
BNA clamp
real-time PCR technology and detection assays
One of the sensitive diagnostic methods for quantitation of changes in
gene expression related to cancer or other diseases is the clamp real-time PCR
technology[40]. The technology can be used with 2’,4’-BNANC
bases to improve the sensitivity of mutation detection due to the fact that the
2’,4’-BNANC bases have the ability to interact with the target
nucleic acids with better affinity and stability in comparison with other
chemically modified nucleotides. Rahman et al also reported that,
compared to other probe technologies, 2’,4’-BNANC probes have proven
to be a sensitive, accurate and robust detection system[27].
Recently, our research group has showed that the results using BNA clamp
technology against EGFR T790M mutation was very promising, where the real-time
PCR detection displayed the highly selective amplification of the target gene
when as little as 0.01% mutated DNA is in the sample analyzed[41].
This result reflects that 2’,4’-BNANC based probes are ideal for
discriminating mutated sequences among wild-type sequences. Similar to the
clamp technology, a bead-based suspension assay was developed using 2',4'-BNANC
probes, and the assay allows quantitative detection of mutations in the human
DNA methyl transferase 3A gene (DNMT3A), whose mutations are found in several
hematologic malignancies[42]. This assay was shown to be a rapid,
easy and reliable method with a sensitivity of 2.5% for different mutant
alleles[42]. This type of assay can be added to the repertoire of
molecular diagnostic techniques with respect to the analysis of biological
markers in genomic research.One of the sensitive diagnostic methods for
quantitation of changes in gene expression related to cancer or other diseases
is the clamp real-time PCR technology[40]. The technology can be
used with 2’,4’-BNANC bases to improve the sensitivity of mutation
detection due to the fact that the 2’,4’-BNANC bases have the
ability to interact with the target nucleic acids with better affinity and
stability in comparison with other chemically modified nucleotides. Rahman et
al also reported that, compared to other probe technologies, 2’,4’-BNANC
probes have proven to be a sensitive, accurate and robust detection system[27].
Recently, our research group has showed that the results using BNA clamp
technology against EGFR T790M mutation was very promising, where the real-time
PCR detection displayed the highly selective amplification of the target gene
when as little as 0.01% mutated DNA is in the sample analyzed[41].
This result reflects that 2’,4’-BNANC based probes are ideal for
discriminating mutated sequences among wild-type sequences. Similar to the
clamp technology, a bead-based suspension assay was developed using 2',4'-BNANC
probes, and the assay allows quantitative detection of mutations in the human
DNA methyl transferase 3A gene (DNMT3A), whose mutations are found in several
hematologic malignancies[42]. This assay was shown to be a rapid,
easy and reliable method with a sensitivity of 2.5% for different mutant
alleles[42]. This type of assay can be added to the repertoire of
molecular diagnostic techniques with respect to the analysis of biological markers
in genomic research.
Conclusion
We have discussed the potential of the new generation BNA. The BNA
technology is very promising mainly because of the great affinity against the
complementary RNA or DNA and the great cellular nuclease resistance. We can
envision that BNA like LNA will be more widely used in antisense
oligonucleotide technology, in the field of molecular diagnostics, and in the
newly emerging field of siRNAs. In addition, we envision that BNA will be
further used as in situ hybridization probes (also known as FISH probes) and
single nucleotide polymorphisms discrimination. BNA’s clear promise has
prompted scientists to produce a diverse set of BNA iterations; for example,
amido-BNA, (S)-cEt-BNA, sulfonamide-BNA, benzylidene acetal-type BNA, and
guanidine-BNA were recently synthesized. Amido-BNA has a similar effect on
antisense knockdown as do other BNAs, and significantly improves cellular
uptake as a salient feature[43]; (S)-cEt-BNA, a constrained
5-methyluracil nucleoside, can serve as a key gapmer unit in antisense
oligonucleosides using relatively short steps in the synthesis process[44];
sulfonamide-BNA enhances nuclease resistance and has a selective effect on the
binding affinity towards single-stranded RNA[45]; benzylidene acetal-type
BNA, a bridged structure that exhibits cleavage by external stimuli, displays
different stability and resistance to enzymatic digestion[46];
guanidine-BNA, a BNA analog with guanidine attached, significantly improves
thermal stability and nuclease resistance and interestingly displays high
binding affinity for single-stranded DNA specifically[47]. As the
efforts to seek better BNA analogs continue, we can easily expect more examples
of modified BNA to emerge. Meanwhile, it is imperative that more investigation
of these new compounds should be required for practical applications. Taken all
together, the technologies using BNA bases have a great potential to become the
next generation methods for disease detection and therapy, and BNA will no
doubt play a central role as essential molecules in these fields.
Acknowledgements
This work is supported by Bio-Synthesis Inc. The authors are deeply
grateful to Edward S. Kim for reviewing this paper.
CONFLICT OF INTERESTS
The Author has no conflicts of interest to declare.
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Peer reviewers: Hongtao, Professor, College of Food Science, South China Agriculture
University, Guangzhou 510642, P.R. China; David Volk, Assistant Professor,
Brown Foundation Institute of Molecular Medicine and Department of Nanomedicine
and Biomedical Engineering, University of Texas Health Science Center at
Houston, Houston, USA.
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