![Difference DNA RNA-EN](https://upload.wikimedia.org/wikipedia/commons/3/37/Difference_DNA_RNA-EN.svg)
Nucleic acid analogues are compounds which are
analogous
Analogy (from Greek ''analogia'', "proportion", from ''ana-'' "upon, according to" lso "against", "anew"+ ''logos'' "ratio" lso "word, speech, reckoning" is a cognitive process of transferring information or meaning from a particular subject ( ...
(structurally similar) to naturally occurring
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
and
DNA, used in medicine and in molecular biology research.
Nucleic acid
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
s are chains of nucleotides, which are composed of three parts: a
phosphate
In chemistry, a phosphate is an anion, salt, functional group or ester derived from a phosphoric acid. It most commonly means orthophosphate, a derivative of orthophosphoric acid .
The phosphate or orthophosphate ion is derived from phospho ...
backbone, a pentose sugar, either
ribose
Ribose is a simple sugar and carbohydrate with molecular formula C5H10O5 and the linear-form composition H−(C=O)−(CHOH)4−H. The naturally-occurring form, , is a component of the ribonucleotides from which RNA is built, and so this compo ...
or
deoxyribose
Deoxyribose, or more precisely 2-deoxyribose, is a monosaccharide with idealized formula H−(C=O)−(CH2)−(CHOH)3−H. Its name indicates that it is a deoxy sugar, meaning that it is derived from the sugar ribose by loss of a hydroxy group. D ...
, and one of four
nucleobase
Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...
s.
An analogue may have any of these altered.
Typically the analogue nucleobases confer, among other things, different base pairing and base stacking properties. Examples include universal bases, which can pair with all four canonical bases, and phosphate-sugar backbone analogues such as
PNA, which affect the properties of the chain (PNA can even form a
triple helix
In the fields of geometry and biochemistry, a triple helix (plural triple helices) is a set of three congruent geometrical helices with the same axis, differing by a translation along the axis. This means that each of the helices keeps the same d ...
).
Nucleic acid analogues are also called
Xeno Nucleic Acid and represent one of the main pillars of
xenobiology
Xenobiology (XB) is a subfield of synthetic biology, the study of synthesizing and manipulating biological devices and systems. The name "xenobiology" derives from the Greek word ''xenos'', which means "stranger, alien". Xenobiology is a form o ...
, the design of new-to-nature forms of life based on alternative biochemistries.
Artificial nucleic acids include
peptide nucleic acid
Peptide nucleic acid (PNA) is an artificially synthesized polymer similar to DNA or RNA.
Synthetic peptide nucleic acid oligomers have been used in recent years in molecular biology procedures, diagnostic assays, and antisense therapies. Due to ...
(PNA),
Morpholino
A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure contains ...
and
locked nucleic acid
A locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), and often referred to as inaccessible RNA, is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The ...
(LNA), as well as
glycol nucleic acid
Glycol nucleic acid (GNA), sometimes also referred to as glycerol nucleic acid, is a nucleic acid similar to DNA or RNA but differing in the composition of its sugar-phosphodiester backbone, using propylene glycol in place of ribose or deoxyribo ...
(GNA),
threose nucleic acid Threose nucleic acid (TNA) is an artificial genetic polymer in which the natural five-carbon ribose sugar found in RNA has been replaced by an unnatural four-carbon threose sugar.Schöning, K. U. ''et al.'' Chemical etiology of nucleic acid structur ...
(TNA) and hexitol nucleic acids (HNA). Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.
In May 2014, researchers announced that they had successfully introduced two new artificial
nucleotides
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules w ...
into bacterial DNA, and by including individual artificial nucleotides in the culture media, were able to passage the bacteria 24 times; they did not create mRNA or proteins able to use the artificial nucleotides. The artificial nucleotides featured 2 fused aromatic rings.
Medicine
Several nucleoside analogues are used as antiviral or anticancer agents. The
viral polymerase incorporates these compounds with non-canonical bases. These compounds are activated in the cells by being converted into nucleotides, they are administered as nucleosides since charged nucleotides cannot easily cross cell membranes.
Molecular biology
![Nucleoside analogues](https://upload.wikimedia.org/wikipedia/commons/3/35/Nucleoside_analogues.svg)
Nucleic acid analogues are used in molecular biology for several purposes:
Investigation of possible scenarios of the origin of life: By testing different analogs, researchers try to answer the question of whether life's use of DNA and RNA was selected over time due to its advantages, or if they were chosen by arbitrary chance;
As a tool to detect particular sequences: XNA can be used to tag and identify a wide range of DNA and RNA components with high specificity and accuracy;
As an enzyme acting on DNA, RNA and XNA substrates - XNA has been shown to have the ability to
cleave and
ligate DNA, RNA and other XNA molecules similar to the actions of RNA
ribozyme
Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonst ...
s;
As a tool with resistance to
RNA hydrolysis RNA hydrolysis is a reaction in which a phosphodiester bond in the sugar-phosphate backbone of RNA is broken, cleaving the RNA molecule. RNA is susceptible to this base-catalyzed hydrolysis because the ribose sugar in RNA has a hydroxyl group at the ...
;
Investigation of the mechanisms used by enzyme;
Investigation of the structural features of nucleic acids.
Backbone analogues
Hydrolysis resistant RNA-analogues
![Morpholino-monomer](https://upload.wikimedia.org/wikipedia/commons/b/b3/Morpholino-monomer.svg)
To overcome the fact that
ribose
Ribose is a simple sugar and carbohydrate with molecular formula C5H10O5 and the linear-form composition H−(C=O)−(CHOH)4−H. The naturally-occurring form, , is a component of the ribonucleotides from which RNA is built, and so this compo ...
's 2' hydroxy group that reacts with the phosphate linked 3' hydroxy group (RNA is too unstable to be used or synthesized reliably), a ribose analogue is used. The most common RNA analogues are 2'-O-methyl-substituted RNA,
locked nucleic acid
A locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), and often referred to as inaccessible RNA, is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The ...
(LNA) or
bridged nucleic acid A bridged nucleic acid (BNA) is a modified RNA nucleotide. They are sometimes also referred to as constrained or inaccessible RNA molecules. BNA monomers can contain a five-membered, six-membered or even a seven-membered bridged structure with a "fi ...
(BNA),
morpholino
A Morpholino, also known as a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO), is a type of oligomer molecule (colloquially, an oligo) used in molecular biology to modify gene expression. Its molecular structure contains ...
, and peptide nucleic acid (
PNA). Although these oligonucleotides have a different backbone sugar or, in the case of PNA, an amino acid residue in place of the ribose phosphate, they still bind to RNA or DNA according to Watson and Crick pairing, but are immune to nuclease activity. They cannot be synthesized enzymatically and can only be obtained synthetically using
phosphoramidite strategy or, for PNA, methods of
peptide synthesis
In organic chemistry, peptide synthesis is the production of peptides, compounds where multiple amino acids are linked via amide bonds, also known as peptide bonds. Peptides are chemically synthesized by the condensation reaction of the carboxyl ...
.
Other notable analogues used as tools
Dideoxynucleotides
Dideoxynucleotides are chain-elongating inhibitors of DNA polymerase, used in the Sanger method for DNA sequencing. They are also known as 2',3' because both the 2' and 3' positions on the ribose lack hydroxyl groups, and are abbreviated as '' ...
are used in
sequencing
In genetics and biochemistry, sequencing means to determine the primary structure (sometimes incorrectly called the primary sequence) of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succ ...
. These nucleoside triphosphates possess a non-canonical sugar, dideoxyribose, which lacks the 3' hydroxyl group normally present in DNA and therefore cannot bond with the next base. The lack of the 3' hydroxyl group terminates the chain reaction as the DNA polymerases mistake it for a regular deoxyribonucleotide. Another chain-terminating analogue that lacks a 3' hydroxyl and mimics
adenosine
Adenosine ( symbol A) is an organic compound that occurs widely in nature in the form of diverse derivatives. The molecule consists of an adenine attached to a ribose via a β-N9-glycosidic bond. Adenosine is one of the four nucleoside building ...
is called
cordycepin
Cordycepin, or 3'-deoxyadenosine, is a derivative of the nucleoside adenosine, differing from the latter by the replacement of the hydroxy group in the 3' position with a hydrogen. It was initially extracted from the fungus ''Cordyceps militaris ...
. Cordycepin is an anticancer drug that targets
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
replication. Another analogue in sequencing is a nucleobase analogue, 7-deaza-GTP and is used to sequence CG rich regions, instead 7-deaza-ATP is called
tubercidin, an antibiotic.
Precursors to the RNA world
RNA may be too complex to be the first nucleic acid, so before the
RNA world
The RNA world is a hypothetical stage in the evolutionary history of life on Earth, in which self-replicating RNA molecules proliferated before the evolution of DNA and proteins. The term also refers to the hypothesis that posits the existence ...
several simpler nucleic acids that differ in the backbone, such as
TNA and
GNA and
PNA, have been offered as candidates for the first nucleic acids.
Base analogues
Nucleobase structure and nomenclature
Naturally occurring bases can be divided into two classes according to their structure:
*
pyrimidine
Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The other ...
s are six-membered heterocyclic with nitrogen atoms in position 1 and 3.
*
purine
Purine is a heterocyclic compound, heterocyclic aromatic organic compound that consists of two rings (pyrimidine and imidazole) fused together. It is water-soluble. Purine also gives its name to the wider class of molecules, purines, which includ ...
s are bicyclic, consisting of a pyrimidine fused to an imidazole ring.
Artificial nucleotides (''Unnatural Base Pairs'' (UBPs) named ''
d5SICS
d5SICS is an artificial nucleoside containing 6-methylisoquinoline-1-thione-2-yl group instead of a base.
It pairs up with dNaM in a hydrophobic interaction. It was not able to be removed by the error-correcting machinery of the ''E. coli'' into ...
UBP'' and ''
dNaM
dNaM is an artificial nucleobase containing a 3-methoxy-2-naphthyl group instead of a natural base.
When it was originally successfully introduced into DNA for replication in an E. coli semi-synthetic organism, it was paired up with d5SICS. For ...
UBP'') have been inserted into bacterial DNA but these genes did not template mRNA or induce protein synthesis. The artificial nucleotides featured two fused aromatic rings which formed a (d5SICS–dNaM) complex mimicking the natural (dG–dC) base pair.
Mutagens
One of the most common base analogs is
5-bromouracil (5BU), the abnormal base found in the mutagenic nucleotide analog BrdU. When a nucleotide containing 5-bromouracil is incorporated into the DNA, it is most likely to pair with adenine; however, it can spontaneously shift into another
isomer
In chemistry, isomers are molecules or polyatomic ions with identical molecular formulae – that is, same number of atoms of each element – but distinct arrangements of atoms in space. Isomerism is existence or possibility of isomers.
Iso ...
which pairs with a different
nucleobase
Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...
,
guanine
Guanine () ( symbol G or Gua) is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine (uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside is called ...
. If this happens during DNA replication, a guanine will be inserted as the opposite base analog, and in the next DNA replication, that guanine will pair with a cytosine. This results in a change in one base pair of DNA, specifically a
transition mutation
In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mi ...
.
Additionally, HNO2, or nitrous acid is a potent mutagen that acts on replicating and non-replicating DNA. It can cause deamination of the amino groups of Adenine, Guanine and Cytosine. Adenine is deaminated to
hypoxanthine
Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids, where it is present in the anticodon of tRNA in the form of its nucleoside inosine. It has a tautomer known as 6-hydroxypurine. Hyp ...
, which base pairs to cytosine instead of thymine. Cytosine is deaminated to uracil, which base pairs with Adenine instead of Guanine. Deamination of Guanine is not mutagenic. Nitrous acid-induced mutations also are induced to mutate back to wild-type using nitrous acid.
Fluorophores
![Aminoallyl Uridine](https://upload.wikimedia.org/wikipedia/commons/0/08/Aminoallyl_Uridine.svg)
Commonly
fluorophore
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
s (such as
rhodamine
Rhodamine is a family of related dyes, a subset of the triarylmethane dyes. They are derivatives of xanthene. Important members of the rhodamine family are Rhodamine 6G, Rhodamine 123, and Rhodamine B. They are mainly used to dye paper and inks ...
or
fluorescein
Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used ...
) are linked to the ring linked to the sugar (in para) via a flexible arm, presumably extruding from the major groove of the helix. Due to low processivity of the nucleotides linked to bulky adducts such as florophores by taq polymerases, the sequence is typically copied using a nucleotide with an arm and later coupled with a reactive fluorophore (indirect labelling):
* amine reactive:
Aminoallyl nucleotide
Aminoallyl nucleotide is a nucleotide with a modified base containing an allylamine. They are used in post-labeling of nucleic acids by fluorescence detection in microarray. They are reactive with N-Hydroxysuccinimide ester group which helps attac ...
contain a primary amine group on a linker that reacts with the amino-reactive dye such as a
cyanine
Cyanines, also referred to as tetramethylindo(di)-carbocyanines are a synthetic dye family belonging to the polymethine group. Although the name derives etymologically from terms for shades of blue, the cyanine family covers the electromagnetic s ...
or
Alexa Fluor
The Alexa Fluor family of fluorescent dyes is a series of dyes invented by Molecular Probes, now a part of Thermo Fisher Scientific, and sold under the Invitrogen brand name. Alexa Fluor dyes are frequently used as cell and tissue labels in fluore ...
dyes, which contain a reactive leaving group, such as a succinimidyl ester (NHS). (base-pairing amino groups are not affected).
* thiol reactive: thiol containing nucleotides reacts with the fluorophore linked to a reactive leaving group, such as a maleimide.
*
biotin
Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', bor ...
linked nucleotides rely on the same indirect labelling principle (+ fluorescent streptavidin) and are used in
Affymetrix
Affymetrix is now Applied Biosystems, a brand of DNA microarray products sold by Thermo Fisher Scientific that originated with an American biotechnology research and development and manufacturing company of the same name. The Santa Clara, Califor ...
DNAchips.
Fluorophores find a
variety of uses in medicine and biochemistry.
Fluorescent base analogues
The most commonly used and commercially available fluorescent base analogue, 2-aminopurine (2-AP), has a high-fluorescence quantum yield free in solution (0.68) that is considerably reduced (appr. 100 times but highly dependent on base sequence) when incorporated into nucleic acids. The emission sensitivity of 2-AP to immediate surroundings is shared by other promising and useful fluorescent base analogues like 3-MI, 6-MI, 6-MAP, pyrrolo-dC (also commercially available), modified and improved derivatives of pyrrolo-dC, furan-modified bases and many other ones (see recent reviews). This sensitivity to the microenvironment has been utilized in studies of e.g. structure and dynamics within both DNA and RNA, dynamics and kinetics of DNA-protein interaction and electron transfer within DNA.
A newly developed and very interesting group of fluorescent base analogues that has a fluorescence quantum yield that is nearly insensitive to their immediate surroundings is the tricyclic cytosine family. 1,3-Diaza-2-oxophenothiazine, tC, has a fluorescence quantum yield of approximately 0.2 both in single- and in double-strands irrespective of surrounding bases. Also the oxo-homologue of tC called tC
O (both commercially available), 1,3-diaza-2-oxophenoxazine, has a quantum yield of 0.2 in double-stranded systems. However, it is somewhat sensitive to surrounding bases in single-strands (quantum yields of 0.14–0.41). The high and stable quantum yields of these base analogues make them very bright, and, in combination with their good base analogue properties (leaves DNA structure and stability next to unperturbed), they are especially useful in fluorescence anisotropy and FRET measurements, areas where other fluorescent base analogues are less accurate. Also, in the same family of cytosine analogues, a FRET-acceptor base analogue, tC
nitro, has been developed. Together with tC
O as a FRET-donor this constitutes the first nucleic acid base analogue FRET-pair ever developed. The tC-family has, for example, been used in studies related to polymerase DNA-binding and DNA-polymerization mechanisms.
Natural non-canonical bases
In a cell, there are several non-canonical bases present: CpG islands in DNA (are often methylated), all eukaryotic mRNA (capped with a methyl-7-guanosine), and several bases of rRNAs (are methylated). Often, tRNAs are heavily modified postranscriptionally in order to improve their conformation or base pairing, in particular in/near the anticodon:
inosine
Inosine is a nucleoside that is formed when hypoxanthine is attached to a ribose ring (also known as a ribofuranose) via a β-N9-glycosidic bond. It was discovered in 1965 in analysis of RNA transferase.
Inosine is commonly found in tRNAs and is e ...
can base pair with C, U, and even with A, whereas thiouridine (with A) is more specific than uracil (with a purine).
Other common tRNA base modifications are pseudouridine (which gives its name to the TΨC loop), dihydrouridine (which does not stack as it is not aromatic), queuosine, wyosine, and so forth. Nevertheless, these are all modifications to normal bases and are not placed by a polymerase.
Base-pairing
Canonical bases may have either a carbonyl or an amine group on the carbons surrounding the nitrogen atom furthest away from the glycosidic bond, which allows them to
base pair
A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA ...
(Watson-Crick base pairing) via hydrogen bonds (amine with ketone, purine with pyrimidine). Adenine and 2-aminoadenine have one/two amine group(s), whereas thymine has two carbonyl groups, and cytosine and guanine are mixed amine and carbonyl (inverted in respect to each other).
The precise reason why there are only four nucleotides is debated, but there are several unused possibilities.
Furthermore, adenine is not the most stable choice for base pairing: in Cyanophage S-2L
diaminopurine (DAP) is used instead of adenine (
host evasion).
Diaminopurine basepairs perfectly with thymine as it is identical to adenine but has an amine group at position 2 forming 3 intramolecular hydrogen bonds, eliminating the major difference between the two types of basepairs (Weak:A-T and Strong:C-G). This improved stability affects protein-binding interactions that rely on those differences.
Other combination include,
* isoguanine and isocytosine, which have their amine and ketone inverted compared to standard guanine and cytosine, (not used probably as tautomers are problematic for base pairing, but isoC and isoG can be amplified correctly with PCR even in the presence of the 4 canonical bases)
* diaminopyrimidine and a xanthine, which bind like 2-aminoadenine and thymine but with inverted structures (not used as xanthine is a deamination product)
However, correct DNA structure can form even when the bases are not paired via hydrogen bonding; that is, the bases pair thanks to hydrophobicity, as studies have shown using DNA
isostere Classical Isosteres are molecules or ions with similar shape and often electronic properties. Many definitions are available. but the term is usually employed in the context of bioactivity and drug development. Such biologically-active compounds co ...
s (analogues with same number of atoms), such as the thymine analogue 2,4-difluorotoluene (F) or the adenine analogue 4-methylbenzimidazole (Z). An alternative hydrophobic pair could be isoquinoline, and the pyrrolo
,3-byridine
Other noteworthy basepairs:
*Several fluorescent bases have also been made, such as the 2-amino-6-(2-thienyl)purine and pyrrole-2-carbaldehyde base pair.
*Metal-coordinated bases, such as pairing between a pyridine-2,6-dicarboxylate (tridentate ligand) and a pyridine (monodentate ligand) through square planar coordination to a central copper ion.
*Universal bases may pair indiscriminately with any other base, but, in general, lower the melting temperature of the sequence considerably; examples include 2'-deoxyinosine (hypoxanthine deoxynucleotide) derivatives, nitroazole analogues, and hydrophobic aromatic non-hydrogen-bonding bases (strong stacking effects). These are used as proof of concept and, in general, are not utilised in degenerate primers (which are a mixture of primers).
*The numbers of possible base pairs is doubled when
xDNA
xDNA (also known as expanded DNA or benzo-homologated DNA) is a size-expanded nucleotide system synthesized from the fusion of a benzene ring and one of the four natural bases: adenine, guanine, cytosine, and thymine. This size expansion produ ...
is considered. xDNA contains expanded bases, in which a benzene ring has been added, which may pair with canonical bases, resulting in four possible base-pairs (8 bases:xA-T,xT-A,xC-G,xG-C, 16 bases if the unused arrangements are used). Another form of benzene added bases is yDNA, in which the base is widened by the benzene.
Metal base-pairs
In metal base-pairing, the
Watson-Crick hydrogen bonds are replaced by the interaction between a metal ion with nucleosides acting as ligands. The possible geometries of the metal that would allow for duplex formation with two
bidentate nucleosides around a central metal atom are:
tetrahedral
In geometry, a tetrahedron (plural: tetrahedra or tetrahedrons), also known as a triangular pyramid, is a polyhedron composed of four triangular faces, six straight edges, and four vertex corners. The tetrahedron is the simplest of all the o ...
,
dodecahedral
In geometry, a dodecahedron (Greek , from ''dōdeka'' "twelve" + ''hédra'' "base", "seat" or "face") or duodecahedron is any polyhedron with twelve flat faces. The most familiar dodecahedron is the regular dodecahedron with regular pentagon ...
, and
square planar
The square planar molecular geometry in chemistry describes the stereochemistry (spatial arrangement of atoms) that is adopted by certain chemical compounds. As the name suggests, molecules of this geometry have their atoms positioned at the corne ...
. Metal-complexing with DNA can occur by the formation of non-canonical base pairs from natural nucleobases with participation by metal ions and also by the exchanging the hydrogen atoms that are part of the Watson-Crick base pairing by metal ions. Introduction of metal ions into a DNA duplex has shown to have potential magnetic, conducting properties, as well as increased stability.
Metal complexing has been shown to occur between natural
nucleobase
Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...
s. A well-documented example is the formation of T-Hg-T, which involves two deprotonated
thymine
Thymine () ( symbol T or Thy) is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nu ...
nucleobases that are brought together by Hg
2+ and forms a connected metal-base pair. This motif does not accommodate stacked Hg
2+ in a duplex due to an intrastrand hairpin formation process that is favored over duplex formation. Two thymines across from each other in a duplex do not form a Watson-Crick base pair in a duplex; this is an example where a Watson-Crick basepair mismatch is stabilized by the formation of the metal-base pair. Another example of a metal complexing to natural nucleobases is the formation of A-Zn-T and G-Zn-C at high pH; Co
+2 and Ni
+2 also form these complexes. These are Watson-Crick base pairs where the divalent cation in coordinated to the nucleobases. The exact binding is debated.
A large variety of artificial nucleobases have been developed for use as metal base pairs. These modified nucleobases exhibit tunable electronic properties, sizes, and binding affinities that can be optimized for a specific metal. For, example a nucleoside modified with a pyridine-2,6-dicarboxylate has shown to bind tightly to Cu
2+, whereas other divalent ions are only loosely bound. The tridentate character contributes to this selectivity. The fourth coordination site on the copper is saturated by an oppositely arranged pyridine nucleobase.
The asymmetric metal base pairing system is orthogonal to the Watson-Crick base pairs. Another example of an artificial nucleobase is that with hydroxypyridone nucleobases, which are able to bind Cu
2+ inside the DNA duplex. Five consecutive copper-hydroxypyridone base pairs were incorporated into a double strand, which were flanked by only one natural nucleobase on both ends. EPR data showed that the distance between copper centers was estimated to be 3.7 ± 0.1 Å, while a natural B-type DNA duplex is only slightly larger (3.4 Å). The appeal for stacking metal ions inside a DNA duplex is the hope to obtain nanoscopic self-assembling metal wires, though this has not been realized yet.
Unnatural base pair (UBP)
An unnatural base pair (UBP) is a designed subunit (or
nucleobase
Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...
) of
DNA which is created in a laboratory and does not occur in nature. In 2012, a group of American scientists led by Floyd Romesberg, a chemical biologist at the
Scripps Research Institute
Scripps Research, previously known as The Scripps Research Institute (TSRI), is a nonprofit American medical research facility that focuses on research and education in the biomedical sciences. Headquartered in San Diego, California, the institu ...
in San Diego, California, published that his team designed an unnatural base pair (UBP).
The two new artificial nucleotides or ''Unnatural Base Pair'' (UBP) were named
d5SICS
d5SICS is an artificial nucleoside containing 6-methylisoquinoline-1-thione-2-yl group instead of a base.
It pairs up with dNaM in a hydrophobic interaction. It was not able to be removed by the error-correcting machinery of the ''E. coli'' into ...
and
dNaM
dNaM is an artificial nucleobase containing a 3-methoxy-2-naphthyl group instead of a natural base.
When it was originally successfully introduced into DNA for replication in an E. coli semi-synthetic organism, it was paired up with d5SICS. For ...
. More technically, these artificial
nucleotides
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules w ...
bearing hydrophobic
nucleobase
Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...
s, feature two fused
aromatic rings
In chemistry, aromaticity is a chemical property of cyclic (ring-shaped), ''typically'' planar (flat) molecular structures with pi bonds in resonance (those containing delocalized electrons) that gives increased stability compared to saturated ...
that form a (d5SICS–dNaM) complex or base pair in DNA.
In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as a
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
containing natural T-A and C-G base pairs along with the best-performing UBP Romesberg's laboratory had designed, and inserted it into cells of the common bacterium ''E. coli'' that successfully replicated the unnatural base pairs through multiple generations.
This is the first known example of a living organism passing along an expanded genetic code to subsequent generations.
This was in part achieved by the addition of a supportive algal gene that expresses a
nucleotide triphosphate transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into ''E. coli'' bacteria.
Then, the natural bacterial replication pathways use them to accurately replicate the
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
containing d5SICS–dNaM.
The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
s which can be encoded by DNA, from the existing 20 amino acids to a theoretically possible 172, thereby expanding the potential for living organisms to produce novel
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s.
Earlier, the artificial strings of DNA did not encode for anything, but scientists speculated they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses.
Transcription of DNA containing unnatural base pair and translation of corresponding mRNA were actually achieved recently. In November 2017, the same team at the
Scripps Research Institute
Scripps Research, previously known as The Scripps Research Institute (TSRI), is a nonprofit American medical research facility that focuses on research and education in the biomedical sciences. Headquartered in San Diego, California, the institu ...
that first introduced two extra nucleobases into bacterial DNA, reported having constructed a semi-synthetic ''E. coli'' bacteria able to make proteins using such DNA. Its DNA contained six different
nucleobase
Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...
s: four canonical and two artificially added, dNaM and dTPT3 (these two form a pair). Also, this bacteria had two corresponding additional RNA bases included in two new codons, additional tRNAs recognizing these new codons (these tRNAs also contained two new RNA bases within their anticodons) and additional amino acids, making the bacteria able to synthesize "unnatural" proteins.
Another demonstration of UBPs were achieved by Ichiro Hirao's group at
RIKEN institute in Japan. In 2002, they developed an unnatural base pair between 2-amino-8-(2-thienyl)purine (s) and pyridine-2-one (y) that functions ''in vitro'' in transcription and translation, for the site-specific incorporation of non-standard amino acids into proteins. In 2006, they created 7-(2-thienyl)imidazo
,5-byridine (Ds) and pyrrole-2-carbaldehyde (Pa) as a third base pair for replication and transcription. Afterward, Ds and 4-
-(6-aminohexanamido)-1-propynyl2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification. In 2013, they applied the Ds-Px pair to DNA aptamer generation by ''in vitro'' selection (SELEX) and demonstrated the genetic alphabet expansion significantly augment DNA aptamer affinities to target proteins.
Orthogonal system
The possibility has been proposed and studied, both theoretically and experimentally, of implementing an orthogonal system inside cells independent of the cellular genetic material in order to make a completely safe system, with the possible increase in encoding potentials.
Several groups have focused on different aspects:
* novel backbones and base pairs as discussed above
* XNA (
Xeno Nucleic Acid) artificial replication/transcription polymerases starting generally from
T7 RNA polymerase
T7 RNA Polymerase is an RNA polymerase from the T7 bacteriophage that catalyzes the formation of RNA from DNA in the 5'→ 3' direction.
Activity
T7 polymerase is extremely promoter-specific and transcribes only DNA downstream of a T7 promot ...
* ribosomes (
16S sequences with altered anti
Shine-Dalgarno sequence allowing the translation of only orthogonal mRNA with a matching altered Shine-Dalgarno sequence)
* novel tRNA encoding non-natural aminoacids. See
Expanded genetic code
An expanded genetic code is an artificially modified genetic code in which one or more specific codons have been re-allocated to encode an amino acid that is not among the 22 common naturally-encoded proteinogenic amino acids.
The key prerequisi ...
See also
*
Biotin
Biotin (or vitamin B7) is one of the B vitamins. It is involved in a wide range of metabolic processes, both in humans and in other organisms, primarily related to the utilization of fats, carbohydrates, and amino acids. The name ''biotin'', bor ...
*
Dark quencher A dark quencher (also known as a dark sucker) is a substance that absorbs excitation energy from a fluorophore and dissipates the energy as ''heat''; while a typical (fluorescent) quencher re-emits much of this energy as ''light''. Dark quenchers a ...
*
Deoxyribozyme
Deoxyribozymes, also called DNA enzymes, DNAzymes, or catalytic DNA, are DNA oligonucleotides that are capable of performing a specific chemical reaction, often but not always catalytic. This is similar to the action of other biological enzymes, ...
*
Expanded genetic code
An expanded genetic code is an artificially modified genetic code in which one or more specific codons have been re-allocated to encode an amino acid that is not among the 22 common naturally-encoded proteinogenic amino acids.
The key prerequisi ...
*
Fluorophore
A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...
*
Genetics
Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar wor ...
*
Molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
*
Nucleic acid
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
*
Nucleobase
Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...
*
Nucleoside
Nucleosides are glycosylamines that can be thought of as nucleotides without a phosphate group. A nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a five-carbon sugar (ribose or 2'-deoxyribose) whereas a nucleotide ...
*
Nucleotide
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules wi ...
*
Oligonucleotide synthesis
Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure (sequence). The technique is extremely useful in current laboratory practice because it provides a rapid and inexpens ...
*
Ribozyme
Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonst ...
*
Synthetic biology
Synthetic biology (SynBio) is a multidisciplinary area of research that seeks to create new biological parts, devices, and systems, or to redesign systems that are already found in nature.
It is a branch of science that encompasses a broad ran ...
*
Xenobiology
Xenobiology (XB) is a subfield of synthetic biology, the study of synthesizing and manipulating biological devices and systems. The name "xenobiology" derives from the Greek word ''xenos'', which means "stranger, alien". Xenobiology is a form o ...
*
xDNA
xDNA (also known as expanded DNA or benzo-homologated DNA) is a size-expanded nucleotide system synthesized from the fusion of a benzene ring and one of the four natural bases: adenine, guanine, cytosine, and thymine. This size expansion produ ...
*
Hachimoji DNA
Hachimoji DNA (from Japanese ''hachimoji'', "eight letters") is a synthetic nucleic acid analog that uses four synthetic nucleotides in addition to the four present in the natural nucleic acids, DNA and RNA. This leads to four allowed base pa ...
*
Artificially Expanded Genetic Information System
Artificially Expanded Genetic Information System (AEGIS) is a synthetic DNA analog experiment that uses some unnatural base pairs from the laboratories of the Foundation for Applied Molecular Evolution in Gainesville, Florida. AEGIS is a NASA-fu ...
(AEGIS)
*
Xeno nucleic acid
References
{{Nucleic acids
Molecular genetics
Nucleic acids
RNA
RNA interference
Gene expression