Editing Base pair (section)

==Unnatural base pair (UBP)==
{{See also|Artificial gene synthesis|Expanded genetic code|Nucleic acid analogue|Synthetic genomics}}
An unnatural base pair (UBP) is a designed subunit (or [[nucleobase]]) of [[DNA]] which is created in a laboratory and does not occur in nature.  DNA sequences have been described which use newly created nucleobases to form a third base pair, in addition to the two base pairs found in nature, A-T ([[adenine]] – [[thymine]]) and G-C ([[guanine]] – [[cytosine]]).  A few research groups have been searching for a third base pair for DNA, including teams led by [[Steven A. Benner]], [[Philippe Marliere]], [[Floyd E. Romesberg]] and [[Ichiro Hirao]].<ref name="Fikes">{{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| vauthors = Fikes BJ |date=May 8, 2014|work=San Diego Union Tribune|access-date=8 May 2014|url-status=dead |archive-url=https://web.archive.org/web/20140509001048/http://www.utsandiego.com/news/2014/may/08/tp-life-engineered-with-expanded-genetic-code/|archive-date=9 May 2014}}</ref> Some new base pairs based on alternative hydrogen bonding, hydrophobic interactions and metal coordination have been reported.<ref>{{cite journal | vauthors = Yang Z, Chen F, Alvarado JB, Benner SA | title = Amplification, mutation, and sequencing of a six-letter synthetic genetic system | journal = Journal of the American Chemical Society | volume = 133 | issue = 38 | pages = 15105–15112 | date = September 2011 | pmid = 21842904 | pmc = 3427765 | doi = 10.1021/ja204910n }}</ref><ref name="Highly">{{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 = 2793–2806 | date = March 2012 | pmid = 22121213 | pmc = 3315302 | doi = 10.1093/nar/gkr1068 }}</ref><ref name="Malyshev PNAS 20120724"/><ref>{{Cite journal| vauthors = Takezawa Y, Müller J, Shionoya M |date=2017-05-05|title=Artificial DNA Base Pairing Mediated by Diverse Metal Ions|journal=Chemistry Letters|language=en|volume=46|issue=5|pages=622–633|doi=10.1246/cl.160985|issn=0366-7022|doi-access=free}}</ref>

In 1989 Steven Benner (then working at the [[ETH Zurich|Swiss Federal Institute of Technology]] in Zurich) and his team led with modified forms of cytosine and guanine into DNA molecules ''in vitro''.<ref>{{cite journal | vauthors = Switzer C, Moroney SE, Benner SA | title = Enzymatic incorporation of a new base pair into DNA and RNA | date = 1989 | journal = J. Am. Chem. Soc. | volume = 111 | issue = 21| pages = 8322–8323 | doi = 10.1021/ja00203a067 }}</ref> The nucleotides, which encoded RNA and proteins, were successfully replicated ''in vitro''. Since then, Benner's team has been trying to engineer cells that can make foreign bases from scratch, obviating the need for a feedstock.<ref name="Ewan">{{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| vauthors = Callaway E |date=May 7, 2014| work=Nature News| publisher=Huffington Post| access-date=8 May 2014}}</ref>

In 2002, Ichiro Hirao's group in Japan developed an unnatural base pair between 2-amino-8-(2-thienyl)purine (s) and pyridine-2-one (y) that functions in transcription and translation, for the site-specific incorporation of non-standard amino acids into proteins.<ref>{{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 | display-authors = 6 | title = An unnatural base pair for incorporating amino acid analogs into proteins | journal = Nature Biotechnology | volume = 20 | issue = 2 | pages = 177–182 | date = February 2002 | pmid = 11821864 | doi = 10.1038/nbt0202-177 | s2cid = 22055476 }}</ref> 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.<ref>{{cite journal | vauthors = Hirao I, Kimoto M, Mitsui T, Fujiwara T, Kawai R, Sato A, Harada Y, Yokoyama S | display-authors = 6 | 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 = 729–735 | date = September 2006 | pmid = 16929319 | doi = 10.1038/nmeth915 | s2cid = 6494156 }}</ref> Afterward, Ds and 4-[3-(6-aminohexanamido)-1-propynyl]-2-nitropyrrole (Px) was discovered as a high fidelity pair in PCR amplification.<ref>{{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 | name-list-style = vanc }}</ref><ref name="Highly"/> 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.<ref>{{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 = 453–457 | date = May 2013 | pmid = 23563318 | doi = 10.1038/nbt.2556 | s2cid = 23329867 }}</ref>

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 designed an unnatural base pair (UBP).<ref name="Malyshev PNAS 20120724">{{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 = 12005–12010 | date = July 2012 | pmid = 22773812 | pmc = 3409741 | doi = 10.1073/pnas.1205176109 | doi-access = free | bibcode = 2012PNAS..10912005M }}</ref>  The two new artificial nucleotides or ''Unnatural Base Pair'' (UBP) were named [[d5SICS]] and [[dNaM]]. More technically, these artificial [[nucleotide]]s bearing hydrophobic [[nucleobase]]s, feature two fused [[Aromatic hydrocarbon|aromatic rings]] that form a (d5SICS–dNaM) complex or base pair in DNA.<ref name="Ewan"/><ref name="NATJ-20140507" /> His team designed a variety of ''in vitro'' or "test tube" templates containing the unnatural base pair and they confirmed that it was efficiently replicated with high fidelity in virtually all sequence contexts using the modern standard ''in vitro'' techniques, namely [[Polymerase chain reaction|PCR amplification of DNA]] and PCR-based applications.<ref name="Malyshev PNAS 20120724"/> Their results show that for PCR and PCR-based applications, the d5SICS–dNaM unnatural base pair is functionally equivalent to a natural base pair, and when combined with the other two natural base pairs used by all organisms, A–T and G–C, they provide a fully functional and expanded six-letter "genetic alphabet".<ref name="NATJ-20140507"/>

In 2014 the same team from the Scripps Research Institute reported that they synthesized a stretch of circular DNA known as 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 ''[[Escherichia coli|E. coli]]'' that successfully replicated the unnatural base pairs through multiple generations.<ref name="Fikes"/> The [[transfection]] did not hamper the growth of the ''E. coli'' cells and showed no sign of losing its unnatural base pairs to its natural [[DNA repair]] mechanisms. This is the first known example of a living organism passing along an expanded genetic code to subsequent generations.<ref name="NATJ-20140507">{{cite journal | vauthors = Malyshev DA, Dhami K, Lavergne T, Chen T, Dai N, Foster JM, Corrêa IR, Romesberg FE | display-authors = 6 | title = A semi-synthetic organism with an expanded genetic alphabet | journal = Nature | volume = 509 | issue = 7500 | pages = 385–388 | date = May 2014 | pmid = 24805238 | pmc = 4058825 | doi = 10.1038/nature13314 | bibcode = 2014Natur.509..385M }}</ref><ref name="Sample">{{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| vauthors = Sample I |date=May 7, 2014|work=The Guardian|access-date=8 May 2014}}</ref> Romesberg said he and his colleagues created 300 variants to refine the design of nucleotides that would be stable enough and would be replicated as easily as the natural ones when the cells divide.  This was in part achieved by the addition of a supportive [[Algae|algal gene]] that expresses a [[Nucleoside triphosphate|nucleotide triphosphate]] transporter which efficiently imports the triphosphates of both d5SICSTP and dNaMTP into ''E. coli'' bacteria.<ref name="NATJ-20140507"/> Then, the natural bacterial replication pathways use them to accurately replicate a [[plasmid]] containing d5SICS–dNaM. Other researchers were surprised that the bacteria replicated these human-made DNA subunits.<ref name = "fox" />

The successful incorporation of a third base pair is a significant breakthrough toward the goal of greatly expanding the number of [[amino acid]]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]]s.<ref name="Fikes"/> The artificial strings of DNA do not encode for anything yet, but scientists speculate they could be designed to manufacture new proteins which could have industrial or pharmaceutical uses.<ref name="Pollack">{{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 | vauthors = Pollack A | date=May 7, 2014| work=New York Times| access-date=8 May 2014}}</ref> Experts said the synthetic DNA incorporating the unnatural base pair raises the possibility of life forms based on a different DNA code.<ref name = "fox">{{cite news| url=https://www.foxnews.com/health/scientists-create-first-living-organism-containing-artificial-dna/| title=Scientists create first living organism containing artificial DNA| date=May 8, 2014| work=The Wall Street Journal |publisher=Fox News| access-date=8 May 2014}}</ref><ref name = "Pollack" />