Nucleic acid analogue
Nucleic acid analogues are compounds which are
, used in medicine and in molecular biology research. Nucleic acids are chains of nucleotides, which are composed of three parts: a phosphate backbone, a pentose sugar, either ribose or deoxyribose, and one of four nucleobases. An analogue may have any of these altered.[1] 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).[2] Nucleic acid analogues are also called xeno nucleic acids and represent one of the main pillars of xenobiology, the design of new-to-nature forms of life based on alternative biochemistries.Artificial nucleic acids include
In May 2014, researchers announced that they had successfully introduced two new artificial
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.[citation needed]
Molecular biology
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;[3]
- 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;[4]
- 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 ribozymes;[3]
- As a tool with resistance to RNA hydrolysis;
- Investigation of the mechanisms used by enzyme; and
- Investigation of the structural features of nucleic acids.
Backbone analogues
Hydrolysis resistant RNA-analogues
Ribose's 2' hydroxy group reacts with the phosphate linked 3' hydroxy group, making RNA too unstable to be used or synthesized reliably. To overcome this, a ribose analogue can be used. The most common RNA analogues are 2'-O-methyl-substituted RNA, locked nucleic acid (LNA) or bridged nucleic acid (BNA), morpholino,[5][6] 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 while being immune to nuclease activity. They cannot be synthesized enzymatically and can only be obtained synthetically using the phosphoramidite strategy or, for PNA, other methods of peptide synthesis.[citation needed]
Other notable analogues used as tools
Precursors to the RNA world
RNA may be too complex to be the first nucleic acid, so before the
Base analogues
Nucleobase structure and nomenclature
Naturally occurring bases can be divided into two classes according to their structure:
- Pyrimidines are six-membered heterocyclic with nitrogen atoms in position 1 and 3.
- Purines are bicyclic, consisting of a pyrimidine fused to an imidazole ring.
Artificial nucleotides (Unnatural Base Pairs (UBPs) named d5SICS UBP and dNaM 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.[7][8][9]
Mutagens
One of the most common base analogs is
Additionally, nitrous acid (HNO2) 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, 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.[citation needed]
Fluorophores
Commonly fluorophores (such as rhodamine or fluorescein) 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 polymerase]s, the sequence is typically copied using a nucleotide with an arm and later coupled with a reactive fluorophore (indirect labelling):
- Amine reactive: aminoallyl nucleotides contain a primary amine group on a linker that reacts with the amino-reactive dye such as cyanine or Alexa Fluor dyes, which contain a reactive leaving group like succinimidyl ester (NHS). Base-pairing amino groups are not affected.
- Thiol reactive: thiol-containing nucleotides react with the fluorophore linked to a reactive leaving group like maleimide.
- Biotin-linked nucleotides rely on the same indirect labelling principle (and fluorescent streptavidin) and are used in Affymetrix 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.[10] 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,[11] pyrrolo-dC (also commercially available),[12] modified and improved derivatives of pyrrolo-dC,[13] furan-modified bases[14] and many other ones (see recent reviews).[15][16][17][18][19] 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.[citation needed]
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.[20][21] Also the oxo-homologue of tC called tCO (both commercially available), 1,3-diaza-2-oxophenoxazine, has a quantum yield of 0.2 in double-stranded systems.[22] 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, tCnitro, has been developed.[23] Together with tCO 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 (often methylated), all eukaryotic mRNA (capped with a methyl-7-guanosine), and several bases of rRNAs (methylated). Often, tRNAs are heavily modified postranscriptionally in order to improve their conformation or base pairing, in particular in or near the anticodon: inosine can base pair with C, U, and even with A, whereas thiouridine (with A) is more specific than uracil (with a purine).[24] 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. [24]
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 (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).[citation needed]
Natural basepairs | |
---|---|
A GC basepair: purine carbonyl/amine forms three intermolecular hydrogen bonds with pyrimidine amine/carbonyl | An AT basepair: purine amine/- forms two intermolecular hydrogen bonds with pyrimidine carbonyl/carbonyl |
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,
- Isoguanine and isocytosine, which have their amine and ketone inverted compared to standard guanine and cytosine. They are 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.[26]
- Diaminopyrimidine and xanthine, which bind like 2-aminoadenine and thymine but with inverted structures. This pair is not used as xanthine is a deamination product.
Unused basepair arrangements | ||
---|---|---|
A DAP-T base: purine amine/amine forms three intermolecular hydrogen bonds with pyrimidine ketone/ketone | An X-DAP base: purine ketone/ketone forms three intermolecular hydrogen bonds with pyrimidine amine/amine | A iG-iC base: purine amine/ketone forms three intermolecular hydrogen bonds with pyrimidine ketone/amine |
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 with DNA isosteres (analogues with same number of atoms) such as the thymine analogue 2,4-difluorotoluene (F) or the adenine analogue 4-methylbenzimidazole (Z).[27] An alternative hydrophobic pair could be isoquinoline and pyrrolo[2,3-b]pyridine[28]
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.[29]
- 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.[30]
- 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 utilized in degenerate primers (which are a mixture of primers).
- The numbers of possible base pairs is doubled when xDNA is considered. xDNA contains expanded bases, in which a benzene ring has been added, which may pair with canonical bases, resulting in four additional possible base-pairs (xA-T, xT-A, xC-G, xG-C) with eight bases (or 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.[31]
Novel basepairs with special properties | ||
---|---|---|
A F-Z base: methylbenzimidazole does not form intermolecular hydrogen bonds with toluene F/F | An S-Pa base: purine thienyl/amine forms three intermolecular hydrogen bonds with pyrrole -/carbaldehyde | An xA-T base: same bonding as A-T |
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
Metal complexing has been shown to occur between natural nucleobases. A well-documented example is the formation of T-Hg-T, which involves two deprotonated thymine nucleobases that are brought together by Hg2+ and forms a connected metal-base pair.[36] This motif does not accommodate stacked Hg2+ in a duplex due to an intrastrand hairpin formation process that is favored over duplex formation.[37] Two thymines across from each other 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.[38]
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 Cu2+, 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.[39] 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 Cu2+ 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).[40] 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
The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of
Another demonstration of UBPs were achieved by Ichiro Hirao's group at
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,[53] with the possible increase in encoding potentials.[54] Several groups have focused on different aspects:
- Novel backbones and base pairs as discussed above;
- XNA artificial replication and transcription polymerases starting generally from T7 RNA polymerase;[55]
- (Shine-Dalgarno sequences allowing the translation of only orthogonal mRNA with a matching altered Shine-Dalgarno sequence;[56]and
- Novel tRNA encoding non-natural aminoacids for an expanded genetic code.
See also
- Biotin
- Dark quencher
- Deoxyribozyme
- Expanded genetic code
- Fluorophore
- Genetics
- Molecular biology
- Nucleic acid
- Nucleobase
- Nucleoside
- Nucleotide
- Oligonucleotide synthesis
- Ribozyme
- Synthetic biology
- Xenobiology
- xDNA
- Hachimoji DNA
- Artificially Expanded Genetic Information System (AEGIS)
- Xeno nucleic acid
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