Editing Denaturation (biochemistry) (section)

== Nucleic acid denaturation ==
{{main|Nucleic acid thermodynamics}}

[[Nucleic acid]]s (including [[RNA]] and [[DNA]]) are [[nucleotide]] polymers synthesized by [[Polymerase|polymerase enzymes]] during either [[transcription (genetics)|transcription]] or [[DNA replication]]. Following 5'-3' synthesis of the backbone, individual [[Nucleobase|nitrogenous bases]] are capable of interacting with one another via [[hydrogen bond]]ing, thus allowing for the formation of higher-order structures. Nucleic acid denaturation occurs when hydrogen bonding between nucleotides is disrupted, and results in the separation of previously [[Annealing (biology)|annealed]] strands. For example, denaturation of DNA due to high temperatures results in the disruption of  [[base pair]]s and the separation of the double stranded helix into two single strands. Nucleic acid strands are capable of re-annealling when "[[Polymerase chain reaction|normal]]" conditions are restored, but if restoration occurs too quickly, the nucleic acid strands may re-anneal imperfectly resulting in the improper pairing of bases.

=== Biologically-induced denaturation ===
[[Image:DNA Denaturation.png|thumb|DNA denaturation occurs when hydrogen bonds between base pairs are disturbed.]]
The [[non-covalent interactions]] between [[Antiparallel (biochemistry)|antiparallel strands]] in DNA can be broken in order to "open" the [[Nucleic acid double helix|double helix]] when biologically important mechanisms such as DNA replication, transcription, [[DNA repair]] or protein binding are set to occur.<ref name="1st">{{cite journal|last2=Destainville|first2=Nicolas|last3=Manghi|first3=Manoel|date=21 January 2015|title=DNA denaturation bubbles: Free-energy landscape and nucleation/closure rates|journal=The Journal of Chemical Physics|volume=142|issue=3|pages=034903|doi=10.1063/1.4905668|pmid=25612729|last1=Sicard|first1=François|arxiv=1405.3867|bibcode=2015JChPh.142c4903S|s2cid=13967558}}</ref> The area of partially separated DNA is known as the denaturation bubble, which can be more specifically defined as the opening of a DNA double helix through the coordinated separation of base pairs.<ref name="1st" />

The first model that attempted to describe the [[Nucleic acid thermodynamics|thermodynamics]] of the denaturation bubble was introduced in 1966 and called the Poland-Scheraga Model. This model describes the denaturation of DNA strands as a function of [[temperature]]. As the temperature increases, the hydrogen bonds between the base pairs are increasingly disturbed and "denatured loops" begin to form.<ref>Lieu, Simon. "The Poland-Scheraga Model." (2015): 0-5. Massachusetts Institute of Technology, 14 May 2015. Web. 25 Oct. 2016.</ref> However, the Poland-Scheraga Model is now considered elementary because it fails to account for the confounding implications of [[Nucleic acid sequence|DNA sequence]], chemical composition, [[stiffness]] and [[torsion (mechanics)|torsion]].<ref>Richard, C., and A. J. Guttmann. "Poland–Scheraga Models and the DNA Denaturation Transition." ''Journal of Statistical Physics'' 115.3/4 (2004): 925-47. Web.</ref>

Recent thermodynamic studies have inferred that the lifetime of a singular denaturation bubble ranges from 1 microsecond to 1 millisecond.<ref name="2nd">{{cite journal|last2=Libchaber|first2=Albert|last3=Krichevsky|first3=Oleg|date=1 April 2003|title=Bubble Dynamics in Double-Stranded DNA|journal=Physical Review Letters|volume=90|issue=13|pages=138101|doi=10.1103/physrevlett.90.138101|pmid=12689326|last1=Altan-Bonnet|first1=Grégoire|s2cid=1427570|bibcode=2003PhRvL..90m8101A}}</ref> This information is based on established timescales of DNA replication and transcription.<ref name="2nd" /> Currently,{{when|date=December 2017}} biophysical and biochemical research studies are being performed to more fully elucidate the thermodynamic details of the denaturation bubble.<ref name="2nd" />

=== Denaturation due to chemical agents ===
[[File:DNA Denaturation by Formamide.png|thumb|Formamide denatures DNA by disrupting the hydrogen bonds between base pairs. Orange, blue, green, and purple lines represent adenine, thymine, guanine, and cytosine respectively. The three short black lines between the bases and the formamide molecules represent newly formed hydrogen bonds.]]
With [[polymerase chain reaction]] (PCR) being among the most popular contexts in which DNA denaturation is desired, heating is the most frequent method of denaturation.<ref name=":02">{{cite journal|date=2014|title=Characterization of denaturation and renaturation of DNA for DNA hybridization|journal=Environmental Health and Toxicology|volume=29|doi=10.5620/eht.2014.29.e2014007|pmid=25234413|pmc=4168728|last1=Wang|first1=X|page=e2014007}}</ref> Other than denaturation by heat, nucleic acids can undergo the denaturation process through various chemical agents such as [[formamide]], [[guanidine]], [[sodium salicylate]], [[dimethyl sulfoxide]] (DMSO), [[propylene glycol]], and [[urea]].<ref name="ReferenceA">{{cite journal|date=1961|title=Denaturation of deoxyribonucleic acid by formamide|volume=51|issue=1|pages=91013–7|last1=Marmur|first1=J|journal=Biochimica et Biophysica Acta|doi=10.1016/0006-3002(61)91013-7|pmid=13767022}}</ref> These chemical denaturing agents lower the melting temperature (T<sub>m</sub>) by competing for hydrogen bond donors and acceptors with pre-existing [[nitrogenous base]] pairs. Some agents are even able to induce denaturation at room temperature. For example, [[Alkalinity|alkaline]] agents (e.g. NaOH) have been shown to denature DNA by changing [[pH]] and removing hydrogen-bond contributing protons.<ref name=":02"/> These denaturants have been employed to make [[Temperature gradient gel electrophoresis|Denaturing Gradient Gel Electrophoresis gel]] (DGGE), which promotes denaturation of nucleic acids in order to eliminate the influence of nucleic acid shape on their [[Gel electrophoresis of nucleic acids|electrophoretic]] mobility.<ref>{{cite web|url=https://www.nationaldiagnostics.com/electrophoresis/article/denaturing-polyacrylamide-gel-electrophoresis-dna-rna|title=Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA|website=Electrophoresis|date=15 August 2011 |publisher=National Diagnostics|access-date=13 October 2016}}</ref>

==== Chemical denaturation as an alternative ====
The [[Optical rotation|optical activity]] (absorption and scattering of light) and hydrodynamic properties ([[Rotational diffusion|translational diffusion]], [[sedimentation coefficient]]s, and [[rotational correlation time]]s) of [[formamide]] denatured nucleic acids are similar to those of heat-denatured nucleic acids.<ref name="ReferenceA"/><ref>{{cite journal|last2=Bustamante|first2=C|last3=Maestre|first3=M|date=1980|title=The Optical Activity of Nucleic Acids and their Aggregates|journal=Annual Review of Biophysics and Bioengineering|volume=9|issue=1|pages=107–141|doi=10.1146/annurev.bb.09.060180.000543|pmid=6156638|last1=Tinoco|first1=I}}</ref><ref>{{cite journal|date=2002|title=Calculation of hydrodynamic properties of small nucleic acids from their atomic structure|journal=Nucleic Acids Research|volume=30|issue=8|pages=1782–8|doi=10.1093/nar/30.8.1782|pmid=11937632|pmc=113193|last1=Fernandes|first1=M}}</ref> Therefore, depending on the desired effect, chemically denaturing DNA can provide a gentler procedure for denaturing nucleic acids than denaturation induced by heat. Studies comparing different denaturation methods such as heating, beads mill of different bead sizes, probe [[sonication]], and chemical denaturation show that chemical denaturation can provide quicker denaturation compared to the other physical denaturation methods described.<ref name=":02"/> Particularly in cases where rapid renaturation is desired, chemical denaturation agents can provide an ideal alternative to heating. For example, DNA strands denatured with [[Alkalinity|alkaline agents]] such as [[Sodium hydroxide|NaOH]] renature as soon as [[Phosphate-buffered saline|phosphate buffer]] is added.<ref name=":02" />

==== Denaturation due to air ====
Small, [[Electronegativity|electronegative]] molecules such as [[nitrogen]] and [[oxygen]], which are the primary gases in [[Atmosphere of Earth|air]], significantly impact the ability of surrounding molecules to participate in [[hydrogen bond]]ing.<ref name=":1">{{cite journal|last2=Schoeffler|first2=G.|last3=McGlynn|first3=S. P.|date=July 1985|title=The effects of selected gases upon ethanol: hydrogen bond breaking by O and N|journal=Canadian Journal of Chemistry|volume=63|issue=7|pages=1864–1869|doi=10.1139/v85-309|last1=Mathers|first1=T. L.|doi-access=free}}</ref> These molecules compete with surrounding hydrogen bond acceptors for hydrogen bond donors, therefore acting as "hydrogen bond breakers" and weakening interactions between surrounding molecules in the environment.<ref name=":1" /> [[Antiparallel (biochemistry)|Antiparellel strands]] in DNA double helices are non-covalently bound by hydrogen bonding between base pairs;<ref>{{cite book|title=Lehninger principles of biochemistry|date=2008|publisher=W.H. Freeman|isbn=9780716771081|edition=5th|location=New York|last1=Cox|first1=David L. Nelson, Michael M.|url-access=registration|url=https://archive.org/details/lehningerprincip00lehn_1}}</ref> nitrogen and oxygen therefore maintain the potential to weaken the integrity of DNA when exposed to air.<ref name="DNA Air">{{cite journal|last2=Schoeffler|first2=G.|last3=McGlynn|first3=S. P.|date=1982|title=Hydrogen-bond breaking by O/sub 2/ and N/sub 2/. II. Melting curves of DNA|doi=10.2172/5693881|last1=Mathers|first1=T. L.|osti=5693881|url=https://digital.library.unt.edu/ark:/67531/metadc1089485/m2/1/high_res_d/5693881.pdf |archive-url=https://web.archive.org/web/20180724122925/https://digital.library.unt.edu/ark:/67531/metadc1089485/m2/1/high_res_d/5693881.pdf |archive-date=2018-07-24 |url-status=live}}</ref> As a result, DNA strands exposed to air require less force to separate and exemplify lower [[Nucleic acid thermodynamics|melting temperatures]].<ref name="DNA Air" />

=== Applications ===
Many laboratory techniques rely on the ability of nucleic acid strands to separate. By understanding the properties of nucleic acid denaturation, the following methods were created:
* [[Polymerase chain reaction|PCR]]
* [[Southern blot]]
* [[Northern blot]]
* [[DNA sequencing]]