nucleic acid analogue

{{main|Nucleoside analogues}}

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.{{cn|date=January 2024}}

File: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;{{cite journal | vauthors = Taylor AI, Pinheiro VB, Smola MJ, Morgunov AS, Peak-Chew S, Cozens C, Weeks KM, Herdewijn P, Holliger P | title = Catalysts from synthetic genetic polymers | journal = Nature | volume = 518 | issue = 7539 | pages = 427–30 | date = February 2015 | pmid = 25470036 | pmc = 4336857 | doi = 10.1038/nature13982 | bibcode = 2015Natur.518..427T }}
• 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;{{cite journal | vauthors = Wang Q, Chen L, Long Y, Tian H, Wu J | title = Molecular beacons of xeno-nucleic acid for detecting nucleic acid | journal = Theranostics | volume = 3 | issue = 6 | pages = 395–408 | year = 2013 | pmid = 23781286 | pmc = 3677410 | doi = 10.7150/thno.5935 }}
• 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;
• As a tool with resistance to RNA hydrolysis;
• Investigation of the mechanisms used by enzyme; and
• Investigation of the structural features of nucleic acids.

File:Morpholino-monomer.svg

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,{{cite journal | vauthors = Summerton J, Weller D | title = Morpholino antisense oligomers: design, preparation, and properties | journal = Antisense & Nucleic Acid Drug Development | volume = 7 | issue = 3 | pages = 187–95 | date = June 1997 | pmid = 9212909 | doi = 10.1089/oli.1.1997.7.187 | s2cid = 19372403 }}{{cite journal | vauthors = Summerton J | title = Morpholino antisense oligomers: the case for an RNase H-independent structural type | journal = Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression | volume = 1489 | issue = 1 | pages = 141–58 | date = December 1999 | pmid = 10807004 | doi = 10.1016/s0167-4781(99)00150-5 }} 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.{{cn|date=January 2024}}

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.{{cn|date=January 2024}}

{{main|RNA world hypothesis}}

It has been suggested that the RNA world may have been preceded by an "RNA-like world" where other nucleic acids with a different backbone, such as GNA, PNA, and TNA existed, however, evidence for this hypothesis been called "tenuous".{{Cite journal |last1=Robertson |first1=M. P. |last2=Joyce |first2=G. F. |date=2012-05-01 |title=The Origins of the RNA World |journal=Cold Spring Harbor Perspectives in Biology |language=en |volume=4 |issue=5 |pages=a003608 |doi=10.1101/cshperspect.a003608 |issn=1943-0264 |pmc=3331698 |pmid=20739415}}

{{multiple image

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{{For|structures of the analogues that may be mentioned in the literature|Simple aromatic ring}}

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.{{cite news |last=Pollack |first=Andrew | name-list-style = vanc |title=Researchers Report Breakthrough in Creating Artificial Genetic Code |url=https://www.nytimes.com/2014/05/08/business/researchers-report-breakthrough-in-creating-artificial-genetic-code.html |date=May 7, 2014 |work=New York Times |access-date=May 7, 2014 }}{{cite journal |last=Callaway |first=Ewen | name-list-style = vanc |title=First life with 'alien' DNA |date=May 7, 2014 |journal=Nature |doi=10.1038/nature.2014.15179 |s2cid=86967999 }}{{cite journal | vauthors = Malyshev DA, Dhami K, Lavergne T, Chen T, Dai N, Foster JM, Corrêa IR, Romesberg FE | title = A semi-synthetic organism with an expanded genetic alphabet | journal = Nature | volume = 509 | issue = 7500 | pages = 385–88 | date = May 2014 | pmid = 24805238 | pmc = 4058825 | doi = 10.1038/nature13314 | bibcode = 2014Natur.509..385M }}

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 which pairs with a different nucleobase, guanine. 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.{{cn|date=January 2024}}

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.{{cn|date=January 2024}}

{{main|Fluorophore}}

File:Aminoallyl uracil.svg

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.

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.{{cite journal | vauthors = Ward DC, Reich E, Stryer L | title = Fluorescence studies of nucleotides and polynucleotides. I. Formycin, 2-aminopurine riboside, 2,6-diaminopurine riboside, and their derivatives | journal = The Journal of Biological Chemistry | volume = 244 | issue = 5 | pages = 1228–37 | date = March 1969 | doi = 10.1016/S0021-9258(18)91833-8 | pmid = 5767305 | doi-access = free }} 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,{{cite journal | vauthors = Hawkins ME | title = Fluorescent pteridine nucleoside analogs: a window on DNA interactions | journal = Cell Biochemistry and Biophysics | volume = 34 | issue = 2 | pages = 257–81 | year = 2001 | pmid = 11898867 | doi = 10.1385/cbb:34:2:257 | s2cid = 12134698 | url = https://zenodo.org/record/1236297 }} pyrrolo-dC (also commercially available),{{cite journal | vauthors = Berry DA, Jung KY, Wise DS, Sercel AD, Pearson WH, Mackie H, Randolph JB, Somers RL | year = 2004 | title = Pyrrolo-dC and pyrrolo-C: fluorescent analogs of cytidine and 2 '-deoxycytidine for the study of oligonucleotides | journal = Tetrahedron Lett. | volume = 45 | issue = 11| pages = 2457–61 | doi=10.1016/j.tetlet.2004.01.108}} modified and improved derivatives of pyrrolo-dC,{{cite journal | vauthors = Wojciechowski F, Hudson RH | title = Fluorescence and hybridization properties of peptide nucleic acid containing a substituted phenylpyrrolocytosine designed to engage Guanine with an additional H-bond | journal = Journal of the American Chemical Society | volume = 130 | issue = 38 | pages = 12574–75 | date = September 2008 | pmid = 18761442 | doi = 10.1021/ja804233g }} furan-modified bases{{cite journal | vauthors = Greco NJ, Tor Y | title = Simple fluorescent pyrimidine analogues detect the presence of DNA abasic sites | journal = Journal of the American Chemical Society | volume = 127 | issue = 31 | pages = 10784–85 | date = August 2005 | pmid = 16076156 | doi = 10.1021/ja052000a }} and many other ones (see recent reviews).{{cite journal | vauthors = Rist MJ, Marino JP | year = 2002 | title = Fluorescent nucleotide base analogs as probes of nucleic acid structure, dynamics and interactions | journal = Curr. Org. Chem. | volume = 6 | issue = 9| pages = 775–93 | doi=10.2174/1385272023373914}}{{cite journal | vauthors = Wilson JN, Kool ET | title = Fluorescent DNA base replacements: Reporters and sensors for biological systems | journal = Organic & Biomolecular Chemistry | volume = 4 | issue = 23 | pages = 4265–74 | date = December 2006 | pmid = 17102869 | doi = 10.1039/b612284c }}{{cite book | last = Wilhelmsson and Tor | title = Fluorescent Analogs of Biomolecular Building Blocks: Design and Applications | publisher = Wiley | location = New Jersey | year = 2016 | isbn = 978-1-118-17586-6 }}{{cite journal | vauthors = Wilhelmsson LM | title = Fluorescent nucleic acid base analogues | journal = Quarterly Reviews of Biophysics | volume = 43 | issue = 2 | pages = 159–83 | date = May 2010 | pmid = 20478079 | doi = 10.1017/s0033583510000090 | s2cid = 10783202 | url = https://research.chalmers.se/en/publication/127915 }}{{cite journal | vauthors = Sinkeldam RW, Greco NJ, Tor Y | title = Fluorescent analogs of biomolecular building blocks: design, properties, and applications | journal = Chemical Reviews | volume = 110 | issue = 5 | pages = 2579–619 | date = May 2010 | pmid = 20205430 | pmc = 2868948 | doi = 10.1021/cr900301e }} 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.{{cn|date=January 2024}}

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.{{cite journal | vauthors = Wilhelmsson LM, Holmén A, Lincoln P, Nielsen PE, Nordén B | year = 2001 | title = A highly fluorescent DNA base analogue that forms Watson-Crick base pairs with guanine | journal = J. Am. Chem. Soc. | volume = 123 | issue = 10| pages = 2434–35 | doi=10.1021/ja0025797| pmid = 11456897 }}{{cite journal | vauthors = Sandin P, Wilhelmsson LM, Lincoln P, Powers VE, Brown T, Albinsson B | title = Fluorescent properties of DNA base analogue tC upon incorporation into DNA – negligible influence of neighbouring bases on fluorescence quantum yield | journal = Nucleic Acids Research | volume = 33 | issue = 16 | pages = 5019–25 | year = 2005 | pmid = 16147985 | pmc = 1201328 | doi = 10.1093/nar/gki790 }} 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.{{cite journal | vauthors = Sandin P, Börjesson K, Li H, Mårtensson J, Brown T, Wilhelmsson LM, Albinsson B | title = Characterization and use of an unprecedentedly bright and structurally non-perturbing fluorescent DNA base analogue | journal = Nucleic Acids Research | volume = 36 | issue = 1 | pages = 157–67 | date = January 2008 | pmid = 18003656 | pmc = 2248743 | doi = 10.1093/nar/gkm1006 }} 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.{{cite journal | vauthors = Börjesson K, Preus S, El-Sagheer AH, Brown T, Albinsson B, Wilhelmsson LM | title = Nucleic acid base analog FRET-pair facilitating detailed structural measurements in nucleic acid containing systems | journal = Journal of the American Chemical Society | volume = 131 | issue = 12 | pages = 4288–93 | date = April 2009 | pmid = 19317504 | doi = 10.1021/ja806944w | url = https://research.chalmers.se/en/publication/92952 }} 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.

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).{{cite journal | vauthors = Rodriguez-Hernandez A, Spears JL, Gaston KW, Limbach PA, Gamper H, Hou YM, Kaiser R, Agris PF, Perona JJ | title = Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position | journal = Journal of Molecular Biology | volume = 425 | issue = 20 | pages = 3888–906 | date = October 2013 | pmid = 23727144 | pmc = 4521407 | doi = 10.1016/j.jmb.2013.05.018 }} 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.

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).{{cn|date=January 2024}}

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.{{cite journal | vauthors = Kirnos MD, Khudyakov IY, Alexandrushkina NI, Vanyushin BF | title = 2-aminoadenine is an adenine substituting for a base in S-2L cyanophage DNA | journal = Nature | volume = 270 | issue = 5635 | pages = 369–70 | date = November 1977 | pmid = 413053 | doi = 10.1038/270369a0 | bibcode = 1977Natur.270..369K | s2cid = 4177449 }} 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 vs 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. 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.{{cite journal | vauthors = Johnson SC, Sherrill CB, Marshall DJ, Moser MJ, Prudent JR | title = A third base pair for the polymerase chain reaction: inserting isoC and isoG | journal = Nucleic Acids Research | volume = 32 | issue = 6 | pages = 1937–41 | date = 2004 | pmid = 15051811 | pmc = 390373 | doi = 10.1093/nar/gkh522 }}
• 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.

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).{{cite journal | vauthors = Taniguchi Y, Kool ET | title = Nonpolar isosteres of damaged DNA bases: effective mimicry of mutagenic properties of 8-oxopurines | journal = Journal of the American Chemical Society | volume = 129 | issue = 28 | pages = 8836–44 | date = July 2007 | pmid = 17592846 | doi = 10.1021/ja071970q }} An alternative hydrophobic pair could be isoquinoline and pyrrolo[2,3-b]pyridine{{cite journal | vauthors = Hwang GT, Romesberg FE | title = Unnatural substrate repertoire of A, B, and X family DNA polymerases | journal = Journal of the American Chemical Society | volume = 130 | issue = 44 | pages = 14872–82 | date = November 2008 | pmid = 18847263 | pmc = 2675700 | doi = 10.1021/ja803833h }}

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.{{cite journal | vauthors = Kimoto M, Mitsui T, Harada Y, Sato A, Yokoyama S, Hirao I | title = Fluorescent probing for RNA molecules by an unnatural base-pair system | journal = Nucleic Acids Research | volume = 35 | issue = 16 | pages = 5360–69 | year = 2007 | pmid = 17693436 | pmc = 2018647 | doi = 10.1093/nar/gkm508 }}
• 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.{{Cite journal|last1=Atwell|first1=Shane|last2=Meggers|first2=Eric|last3=Spraggon|first3=Glen|last4=Schultz|first4=Peter G.|date=Dec 2001|title=Structure of a Copper-Mediated Base Pair in DNA|url=https://pubs.acs.org/doi/10.1021/ja011822e|journal=Journal of the American Chemical Society|language=en|volume=123|issue=49|pages=12364–12367|doi=10.1021/ja011822e|pmid=11734038|issn=0002-7863}}
• 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.{{cite journal | vauthors = Liu H, Gao J, Lynch SR, Saito YD, Maynard L, Kool ET | title = A four-base paired genetic helix with expanded size | journal = Science | volume = 302 | issue = 5646 | pages = 868–71 | date = October 2003 | pmid = 14593180 | doi = 10.1126/science.1088334 | bibcode = 2003Sci...302..868L | s2cid = 37244007 }}

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.{{cite journal | vauthors = Wettig SD, Lee JS | year = 2003 | title = Thermodynamic investigation of M-DNA: a novel metal ion–DNA complex | journal =Journal of Inorganic Biochemistry| volume = 94 | issue =1–2 | pages = 94–99|doi=10.1016/S0162-0134(02)00624-4 | pmid = 12620678 }} Introduction of metal ions into a DNA duplex has shown to have potential magnetic{{cite journal | vauthors = Zhang HY, Calzolari A, Di Felice R | title = On the magnetic alignment of metal ions in a DNA-mimic double helix | journal = The Journal of Physical Chemistry B | volume = 109 | issue = 32 | pages = 15345–48 | date = August 2005 | pmid = 16852946 | doi = 10.1021/jp052202t }} or conducting properties,{{cite journal | vauthors = Aich P, Skinner RJ, Wettig SD, Steer RP, Lee JS | title = Long range molecular wire behaviour in a metal complex of DNA | journal = Journal of Biomolecular Structure & Dynamics | volume = 20 | issue = 1 | pages = 93–98 | date = August 2002 | pmid = 12144356 | doi = 10.1080/07391102.2002.10506826 | s2cid = 41568646 }} as well as increased stability.{{cite journal | vauthors = Clever GH, Polborn K, Carell T | year = 2005 | title = Ein hochgradig DNA-Duplex-stabilisierendes Metall-Salen-Basenpaar| journal = Angew. Chem. Int. Ed. | volume = 117 | issue = 44| pages = 7370–74 | doi=10.1002/ange.200501589| bibcode = 2005AngCh.117.7370C }}

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.{{cite journal | vauthors = Buncel E, Boone C, Joly H, Kumar R, Norris AR | title = Metal ion-biomolecule interactions. XII. 1H and 13C NMR evidence for the preferred reaction of thymidine over guanosine in exchange and competition reactions with mercury (II) and methylmercury (II) | journal = Inorg. Biochem. | year = 1985 | volume = 25 | pages = 61–73 | doi = 10.1016/0162-0134(85)83009-9 }} This motif does not accommodate stacked Hg²⁺ in a duplex due to an intrastrand hairpin formation process that is favored over duplex formation.{{cite journal | vauthors = Ono A, Togashi H | title = Highly selective oligonucleotide-based sensor for mercury(II) in aqueous solutions | journal = Angewandte Chemie | volume = 43 | issue = 33 | pages = 4300–02 | date = August 2004 | pmid = 15368377 | doi = 10.1002/anie.200454172 }} 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; Co²⁺ and Ni²⁺ also form these complexes. These are Watson-Crick base pairs where the divalent cation in coordinated to the nucleobases. The exact binding is debated.{{cite journal | vauthors = Meggers E, Holland PL, Tolman WB, Romesberg FE, Schultz PG | title = A Novel Copper-Mediated DNA Base Pair | journal= J. Am. Chem. Soc. | year=2000 | volume = 122 | issue = 43| pages = 10714–15 | doi=10.1021/ja0025806}}

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.{{cite journal | vauthors = Lee JS, Latimer LJ, Reid RS | title = A cooperative conformational change in duplex DNA induced by Zn2+ and other divalent metal ions | journal = Biochemistry and Cell Biology | volume = 71 | issue = 3–4 | pages = 162–68 | date = 1993 | pmid = 8398074 | doi = 10.1139/o93-026 }} 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).{{cite journal | vauthors = Tanaka K, Tengeiji A, Kato T, Toyama N, Shionoya M | title = A discrete self-assembled metal array in artificial DNA | journal = Science | volume = 299 | issue = 5610 | pages = 1212–13 | date = February 2003 | pmid = 12595687 | doi = 10.1126/science.1080587 | bibcode = 2003Sci...299.1212T | s2cid = 22413126 }} 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.

{{Main|Base pair#Unnatural base pair (UBP)|l1=Unnatural base pair}}

An unnatural base pair (UBP) is a designed subunit (or nucleobase) of DNA that 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 in San Diego, California, published that his team had designed two unnatural base pairs

named d5SICS and dNaM.{{cite journal | vauthors = Malyshev DA, Dhami K, Quach HT, Lavergne T, Ordoukhanian P, Torkamani A, Romesberg FE | title = Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 30 | pages = 12005–10 | date = July 2012 | pmid = 22773812 | pmc = 3409741 | doi = 10.1073/pnas.1205176109 | bibcode = 2012PNAS..10912005M | doi-access = free }} More technically, these artificial nucleotides bearing hydrophobic nucleobases feature two fused aromatic rings that form a d5SICS–dNaM complex or base pair in DNA.{{cite news| url=http://www.huffingtonpost.com/2014/05/07/living-organism-artificial-dna_n_5283095.html |title=Scientists Create First Living Organism With 'Artificial' DNA| last=Callaway| first=Ewan | name-list-style = vanc |date=May 7, 2014| work=Nature News| publisher=Huffington Post| access-date=8 May 2014}} In 2014, the same team reported that they had synthesized a plasmid 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, which successfully replicated the unnatural base pairs through multiple generations.{{cite news| url=http://www.utsandiego.com/news/2014/may/08/tp-life-engineered-with-expanded-genetic-code/| title=Life engineered with expanded genetic code| last=Fikes| first=Bradley J.| name-list-style = vanc | date=May 8, 2014 |work=San Diego Union Tribune| access-date=8 May 2014}} This is the first known example of a living organism passing along an expanded genetic code to subsequent generations.{{cite news| url=https://www.theguardian.com/world/2014/may/07/living-organism-pass-down-artificial-dna-us-scientists| title=First life forms to pass on artificial DNA engineered by US scientists| last=Sample|first=Ian| name-list-style = vanc |date=May 7, 2014|work=The Guardian|access-date=8 May 2014}} 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 containing d5SICS–dNaM.{{cn|date=January 2024}}

The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of amino acids 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 proteins. 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.{{cite news| url=https://www.nytimes.com/2014/05/08/business/researchers-report-breakthrough-in-creating-artificial-genetic-code.html?hpw&rref=business&_r=0| title=Scientists Add Letters to DNA's Alphabet, Raising Hope and Fear |last=Pollack| first=Andrew | name-list-style = vanc | date=May 7, 2014| work=New York Times| access-date=8 May 2014}} Transcription of DNA containing unnatural base pairs and translation of corresponding mRNA were actually achieved recently. In November 2017, the same team at the Scripps Research Institute 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 nucleobases: four canonical and two artificially added, dNaM and dTPT3 (these two form a pair). The bacteria had two corresponding 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, enabling the bacteria to synthesize "unnatural" proteins.{{cite journal | doi = 10.1038/nature24659 | volume=551 | title=A semi-synthetic organism that stores and retrieves increased genetic information | year=2017 | journal=Nature | pages=644–47 | vauthors=Zhang Y, Ptacin JL, Fischer EC, Aerni HR, Caffaro CE, San Jose K, Feldman AW, Turner CR, Romesberg FE| issue=7682 | pmid=29189780 | pmc=5796663 | bibcode=2017Natur.551..644Z }}[https://www.bbc.com/news/science-environment-42167569 'Unnatural' microbe can make proteins]. BBC News. 29 November 2017.

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.{{cite journal | vauthors = Hirao I, Ohtsuki T, Fujiwara T, Mitsui T, Yokogawa T, Okuni T, Nakayama H, Takio K, Yabuki T, Kigawa T, Kodama K, Yokogawa T, Nishikawa K, Yokoyama S | title = An unnatural base pair for incorporating amino acid analogs into proteins | journal = Nature Biotechnology | volume = 20 | issue = 2 | pages = 177–82 | date = February 2002 | pmid = 11821864 | doi = 10.1038/nbt0202-177 | s2cid = 22055476 }} In 2006, they created 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and pyrrole-2-carbaldehyde (Pa) as a third base pair for replication and transcription.{{cite journal | vauthors = Hirao I, Kimoto M, Mitsui T, Fujiwara T, Kawai R, Sato A, Harada Y, Yokoyama S | title = An unnatural hydrophobic base pair system: site-specific incorporation of nucleotide analogs into DNA and RNA | journal = Nature Methods | volume = 3 | issue = 9 | pages = 729–35 | date = September 2006 | pmid = 16929319 | doi = 10.1038/nmeth915 | s2cid = 6494156 }} Afterward, Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification.{{cite journal | vauthors = Kimoto M, Kawai R, Mitsui T, Yokoyama S, Hirao I | title = An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules | journal = Nucleic Acids Research | volume = 37 | issue = 2 | pages = e14 | date = February 2009 | pmid = 19073696 | pmc = 2632903 | doi = 10.1093/nar/gkn956 }}{{cite journal | vauthors = Yamashige R, Kimoto M, Takezawa Y, Sato A, Mitsui T, Yokoyama S, Hirao I | title = Highly specific unnatural base pair systems as a third base pair for PCR amplification | journal = Nucleic Acids Research | volume = 40 | issue = 6 | pages = 2793–806 | date = March 2012 | pmid = 22121213 | pmc = 3315302 | doi = 10.1093/nar/gkr1068 }} 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.{{cite journal | vauthors = Kimoto M, Yamashige R, Matsunaga K, Yokoyama S, Hirao I | title = Generation of high-affinity DNA aptamers using an expanded genetic alphabet | journal = Nature Biotechnology | volume = 31 | issue = 5 | pages = 453–57 | date = May 2013 | pmid = 23563318 | doi = 10.1038/nbt.2556 | s2cid = 23329867 }}

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,Schmidt M. [http://www.markusschmidt.eu/pdf/Xenobiology-Schmidt_Bioessays_201004.pdf "Xenobiology: a new form of life as the ultimate biosafety tool"] Bioessays Vol 32(4):322–31 with the possible increase in encoding potentials.{{cite journal | vauthors = Herdewijn P, Marlière P | title = Toward safe genetically modified organisms through the chemical diversification of nucleic acids | journal = Chemistry & Biodiversity | volume = 6 | issue = 6 | pages = 791–808 | date = June 2009 | pmid = 19554563 | doi = 10.1002/cbdv.200900083 | s2cid = 8572188 }}

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;{{cite journal | vauthors = Shinkai A, Patel PH, Loeb LA | title = The conserved active site motif A of Escherichia coli DNA polymerase I is highly mutable | journal = The Journal of Biological Chemistry | volume = 276 | issue = 22 | pages = 18836–42 | date = June 2001 | pmid = 11278911 | doi = 10.1074/jbc.M011472200 | doi-access = free }}
• (16S ribosomal sequences with altered anti-Shine-Dalgarno sequences allowing the translation of only orthogonal mRNA with a matching altered Shine-Dalgarno sequence;{{cite journal | vauthors = Rackham O, Chin JW | title = A network of orthogonal ribosome x mRNA pairs | journal = Nature Chemical Biology | volume = 1 | issue = 3 | pages = 159–66 | date = August 2005 | pmid = 16408021 | doi = 10.1038/nchembio719 | s2cid = 37181098 }} and
• Novel tRNA encoding non-natural aminoacids for an expanded genetic code.

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• 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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