Nucleic acid analogues

Not to be confused with degenerate bases.

For phosphoramidite synthesis of nucleic acids, see Oligonucleotide synthesis.

RNA with its nucleobases to the left and DNA to the right.

Nucleic acid analogues are compounds structurally similar (analog) to naturally occurring RNA and DNA, used in medicine and in molecular biology research. Nucleic acids are chains of nucleotides, which are composed of three parts: a phosphate backbone, a pucker-shaped pentose sugar, either ribose or deoxyribose, and one of four nucleobases. An analogue may have any of these altered. Typically the analogue nucleobases confer, among other things, different base pairing and base stacking proprieties. Examples include universal bases, which can pair with all four canon bases, and phosphate-sugar backbone analogues such as PNA, which affect the properties of the chain (PNA can even form a triple helix).^[1]

Artificial nucleic acids include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.

Medicine

Main article: Nucleoside analogues

Several nucleoside analogues are used as antiviral or anticancer agents. The viral polymerase incorporates these compounds with non-canon 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

Common changes in nucleotide analogues

Nucleic acid analogues are used in molecular biology for several purposes:

• As a tool to detect particular sequences
• As a tool with resistance to RNA hydrolysis
• As a tool for another purpose, such as sequencing
• Naturally occurring, such as in tRNA
• Investigation of the mechanisms used by enzyme, such as an Enzyme inhibitor
• Investigation of possible scenarios of the origin of life
• Investigation of the structural features of nucleic acids
• Investigation of the possible alternatives to the natural system in Synthetic biology

Backbone analogues

Hydrolysis resistant RNA-analogues

Chemical structure of Morpholino

To overcome the fact that ribose'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 (LNA), morpholino^[2][3], 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.

Other notable analogues used as tools

Dideoxynucleotides are used in sequencing . 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 is called cordycepin. Cordycepin is an anticancer drug that targets RNA 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

Main article: RNA world hypothesis

RNA may be too complex to be the first nucleic acid, so before the RNA world 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

Main article: Nucleobase

Natural bases are divided into two classes depending on their structure: pyrimidine (an heterocyclic aromatic six-membered ring with nitrogen atoms in position 1 and 3) and purine (a pyrimidine (numeration inverted) fused with an imidazole ring, a five-membered ring with 2 nitrogen atoms separated by one carbon (meta), 7,9). Their main proprieties are base pairing, resulting form 2 or 3 hydrogen bonds between ketone (electron withdrawing group,ei. more negatively charged) and amino (electron releasing group,ei. more positively charged) functional groups, and base stacking, caused by the attraction of the delocalized π electron clouds of the aromatic ring structure.

Purine	Pyrimidine

For structures of the analogues that may be mentioned in the literature, see Simple aromatic ring.

Fluorophores

Main article: Fluorophore

Structure of aminoallyl-uridine

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 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 contain a primary amine group on a linker that reacts with the amino-reactive dye such as a cyanine or Alexa Fluor 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 linked nucleotides rely on the same indirect labelling principle (+ 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.^[4] 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,^[5] pyrrolo-dC (also commercially available),^[6] modified and improved derivatives of pyrrolo-dC^[7], furan-modified bases^[8] and many other ones (see recent reviews).^{[9][10][11][12]} This sensitivity to the microenvironment have 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.^[13][14] 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.^[15] 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.^[16] 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-canon bases

In a cell, there are several noncanon 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 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 ketone 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 ketone groups, and cytosine and guanine are mixed amine and ketone (inverted in respect to each other).

Natural basepairs
A GC basepair: purine ketone/amine forms three intermolecular hydrogen bonds with pyrimidine amine/ketone	An AT basepair: purine amine/- forms two intermolecular hydrogen bonds with pyrimidine ketone/ketone

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).^[17] 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 canon bases)^[18]
• diaminopyrimidine and a xanthine, which bind like 2-aminoadenine and thymine but with inverted structures (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-DAY 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 using DNA isosteres (analogues with same number of atoms), such as the thymine analogue 2,4-difluorotoluene (F) or the adenine analogue 4-methylbenzimidazole (Z).^[19] An alternative hydrophobic pair could be isoquinoline, and the pyrrolo[2,3-b]pyridine^[20]

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.^[21]
• Metal coordinated bases, such as two 2,6-bis(ethylthiomethyl)pyridine (SPy) with a silver ion or pyridine-2,6-dicarboxamide (Dipam) and a mondentate pyridine (Py) with a copper ion.^[22]
• 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 is considered. xDNA contains expanded bases, in which a benzene ring has been added, which may pair with canon 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.^[23]

Novel basepairs with special proprieties
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 bidentate nucleosides around a central metal atom are: tetrahedral, dodecahedral, and square planar. 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.^[24] Introduction of metal ions into a DNA duplex has shown to have potential magnetic,^[25] conducting properties,^[26] as well as increased stability.^[27]

Metal complexing has been shown to occur between natural nucleobases. A well-documented example is the formation of T-Hg-T, which involves two deportonated thymine nucleobases that are brought together by Hg²⁺ and forms a connected metal-base pair.^[28] This motif does not accommodate stacked Hg²⁺ in a duplex due to an intrastrand hairpin formation process that is favored over duplex formation.^[29] 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.^[30]

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.^[31] 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).^[32] 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.

Orthogonal system

It has been proposed and studied both theoretically and experimentally the possibility of implementing an orthogonal system inside cells independent of the cellular genetic material in order to make a completely safe system^[33], with the possible increase in encoding potentials^[34] Several groups have focused on different aspects:

• novel backbones and base pairs as discussed above
• XNA replication/transcription polymerases starting generally from T7 RNA polymerase^[35]
• ribosomes (16S sequences with altered anti Shine-Dalgarno sequence allowing the translation of only orthogonal mRNA with a matching altered Shine-Dalgarno sequence)^[36]
• novel tRNA encoding non-natural aminoacids. See Expanded genetic code

See also

• Molecular biology
• Genetics
• Synthetic biology
• Oligonucleotide synthesis
• Expanded genetic code
• Nucleobase, nucleoside, nucleotide, and nucleic acid
• Biotin, fluorophore and dark quencher
• Ribozyme

References

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