Triplex Forming Oligonucleotides Dna Synthesizing Biology Essay


Triplex Forming Oligonucleotides (TFOs) are synthesized to target DNA in order to control or modify the gene expression during transcription, which would result in decrease in the mRNA (messenger RNA) concentration for an increase period of time. A gene is made up strands of DNA, which forms the functional part of gene. The information for specific proteins is stored in DNA and the transcription of information takes place from DNA to mRNA. The base sequence of mRNA enters the cytoplasm where it binds to amino acids to form specific proteins and this process is called 'translation' .This transcription and translation of gene, when over-expressed, produces abnormal proteins which cause different diseases such as cancer. The size-expanded DNA are prepared in order replicate these genes responsible for such proteins by different routes of nucleic acid targeting. The researchers are working on new design strategies with a different base analogue to target the duplex DNA. There are also complementary antisense oilgoneucleotides, which are single strands of DNA that act as a complementary for the chosen gene sequence on mRNA and inhibit protein translation of some of the mRNA strands by attaching to them. But as antisense DNA can be used to target complementary sequence of RNA, this DNA and RNA combination can be degraded during the binding mechanism. The gene specification and specific targeting through gene strategy and antisense oligonucleotides have shown prospects for future studies regarding the expanded DNA through base-modified oligonucleotide analogues, in order to synthesize analogues with increase affinity and nucleic acid targeting.

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Mahato,R.I (2007)

In this research project, the synthesises of a size-expanded DNA by stretching a base pair will be carried out .The base which is being selected to stretch is one of the most important base pair of DNA which is Adenine. DNA consist of 2 base pairs, first the adenine and thymine and the other base pair is guanine and cytosine .This research would focus on the synthesis of pyridine-stretched adenine phosphoramidite oligonucleotide with the help of an intermediate which is then incorporated in DNA base pair and the it would be synthesized by solid support automated DNA synthesis. The ability of stretched adenine nucleoside to form highly stable TFOs with duplex DNA would be studied and the characterization and efficiency of this triplex will be done under UV thermal melting studies. The strategy in this project is to design a base pair of Stretched Adenine with a heterocyclic ring so that stable and efficient TFOs of duplex DNA is synthesized which has affinity of modifying gene expression and gene transcription. By doing this research it would be further carried out in forming a new genetic system with newly synthesized expanded DNA which can function normally. In addition to this research, the fluorescence properties which a synthesized expanded DNA inherits by conjugation through benzo-fusion will be examined. This fluorescence property is useful for probing DNA and RNA hybridization and protein DNA interactions. This property is not present in natural DNA .The assay of fluorescence is done on these expanded DNA by involving quenching, time-resolved fluorescence test, static fluorescence energy transfer and other fluorescence base assay, as this fluorescence property will evaluate the biophysical and biochemical changes taking place in the helix of DNA. Davis, M.L et al (2008)


In recent times, research on size-expanded DNA has increased and has evolved many positive outcomes about their use in modification of natural DNA by incorporating the stretched base pair in DNA ,in order to change the gene expression .The stability of the expanded DNA is not always the same in each synthesis of different analogues of base pairs, this is due to the difference in chemical structure of the expanded DNA as the stretched base pairs of DNA are smaller in size as compared to the natural DNA. The expanded DNA has more carbon-carbon bonds in between which help them to be synthesized prebioticaly easier. The structural complexity in expanded DNA is more as compared to natural DNA, but scientists synthesize size expanded DNA as it provides advantage to the natural DNA in their structural base assembly in a primordial.

One of the most important aims on researching the size-expanded DNA is to study the strategy to design a non-natural genetic system with known bio-mimetic chemistry, in which the small molecules and biochemical pathways act in a similar manner to the organisms having natural molecules and biochemical pathways. As DNA does not comprise of a genetic system itself, it is the functional encoding part of the genetic system .Therefore in order to make a new genetic system function it is necessary to have a monomeric building blocks and polymerase enzymes which are responsible for replication. By this it is possible to form a design with help of an expanded molecule of DNA analogue to form a new genetic system.

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In size-expanded DNA synthesis there are high amount of studies still required regarding the extent of bio-mimicry of DNA .The replication of the size-expanded with natural DNA is still to be discovered with positive results.

The transcription of the expanded DNA which is stopped during the stretched base analogue synthesis and also their functioning as a gene transcriptor when they are incorporated in natural DNA to modify the DNA structure are still studied more over for accurate results. The researchers are also focusing on the use of expanded DNA in the field of biotechnology and nanotechnology. All these studies about size-expanded DNA and their base pair are still to be discovered in order to increase the application of stable size-expanded DNA in the field of medicine.

Mahato, R.I (2007) ; Krueger, A.T et al (2007)

Size-expanded DNA takes place by modifications in the base pairs of DNA and the DNA bases are Adenine, Guanine, Cytosine and Thymine. Thus the incorporation of bicyclic, tricyclic and tetracyclic base analogues of oligonucleotides are used with the base pairs of DNA to stretch them in order to increase the gene specification sequences and to improve the nucleic acid targeting of DNA.

Davis, M.L et al (2008)

The start of stretching the DNA base pairs was carried out initially by Leonard in 1974.He synthesized an analogue called adenine ribonucleoside (1) by incorporating benzene ring to stretch out the adenine base

Adenine ribonucleoside (1)

This benzo-adenosine substrate was then used as a substrate for different ATP enzymatic action and AMP protein kinase. Following this, another analogue called 2'deoxy variant to benzo-A from ribonucleoside was synthesized. After this research Leonard observed that these analogues are bigger in size as compared to natural DNA or RNA helix. However it is observed that only some examples of the bases being bigger in size are there compared to the natural ones in DNA.

Then studies were carried out to prepare Benzo-fused DNA bases which displayed the point of attachment at natural points by Moreau and by Saito, thus expressing that base with natural partners were likely to take on the normal, instead of having the stretched geometry .These studies are more related to our research strategy. An adenine expanded molecule was prepared to observe its potential pairing sizes called the thiophene-fused adenine like nucleobase (2). However in this case this molecule was not further studied by incorporating it in to DNA.

After these studies, further research was carried out by Matsudda who examined the pairing properties by preparing DNA bases consisting of extra rings (3) in which few of them were prepared to have 4 hydrogen bonds in each pair, keeping the original structure of DNA unchanged.

(2) (3)

Krueger,A.T et al (2007)

Recent research and studies are being carried out to synthesize new size-expanded DNA, by incorporating different heterocyclic rings in base pairs of natural DNA to modify the stable genetic system, which have the affinity and stability to function similarly as natural DNA and to modify the gene sequencing and expression. However there are still different characteristics to be determined in size expanded DNA bases with different base pair modifications while targeting in DNA, the researchers are studying different properties with new research work on the expanded DNA analogue.


Pyridine stretched nucleosides (PSN) are tricyclic analogues of purine bases containing pyridine ring in between the pyrimidine and imidazole rings. PSN were synthesized for their incorporation in TFOs to observe their effects on stability and affinity. There were three pyridine stretched 2'deoxynucleosides analogues which are stretched 2'deoxyadenine (Str A),stretched 2'deoxydiaminopurine (Str D) and 2'deoxyhypoxanthine (Str H).To synthesize these pyridine stretched bases, an intermediate was formed which is imidazo[4,5-b]pyridine.This intermediate was formed from a silver crystalline salt , 4-(5)nitroimidazole through glycolysation by undergoing three isolation steps. The salt 4-(5) nitroimidazole was glycosylated with 1'chloro-2'deoxyy sugar which yielded two β isomers 4-nitro-regioisomer and 5-nitro-regiosiomer in a required ratio of 2:1.The 4-5 nitroimidazole was again glycosylated with lithium to yield two more α isomers. Then 5-nitroimidazole was treated in a catalytic reaction through which an amine was yielded and was immediately reacted with ethoxymethylene malononitrile (EMMN) which is C-addition elimination product. This (EMMN) was further treated with methaloonic NaOH under reflux action to yield the intermediate imidazo [4-5-b] pyridine. This intermediate was then further treated on with exposure to high amount of diethoxy methyl acetate in a form of suspension, under reflux conditions for about 2hrs. O-acetylimidate was obtained from evaporation. It appeared to be brown oil which was then treated with methalonic ammonia for 16hrs to remove the solvent and to yield formamidine. This was then treated with hydro acetonitrile to form a crystalline product and after treating this crystalline powder with H2O it was yielded as Str A. Then Str D was yielded by treating Guanidine dihydrochloride with NaOME in MeOH to form a free base .This free base was mixed equivalent portion of imidazo 4-5-b pyridine and was heated for 42 hrs at 145 °c temperature. The crystalline product was filtered with water first and then with EtOH. After this washing Str D was identified. Str H was obtained by the hydrolysis of 4-5-b-pyridine with H2O2 and NH4OH which yielded an amide which was treated with ethyl format and ethanolic acid at a reflux temperature. The remaining crystalline product was then filtered with H2O and Str H was identified.

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Clayton,R. Et al (2002)

The recent research's on the size expanded DNA has been done and have given some positive results. A research carried out by using a photochemical metal Ruthenium which is lanthanide metal and attaching it in to a specific site at oligoncleotides. Ru(bpy)32+, a photochemical complex was synthesized into a phosphoramidite by reacting Ru(bpy)2Cl2 with 4-methyl-2,2'-bipyridine- 4'-carbonylethanolamide.This reaction yielded tris-bipyridine ruthenium complex .From this complex, PF6+ was isolated which is soluble in organic solvents. Then this salt was treated with 2-cyanoethylchloro-N, Ndiisopropylphosphoramidite dry CH3CN in order to yeild methallo-phosphoramidite. This yielded salt was then introduced when ruthenium phosphoramidite was added when it under goes reflux using automated solid phase DNA synthesis, the Ru(bpy)32+ phosphoramidite was incorporated at 5'-terminus in DNA oligonuctleotide to form (Ru) trisdiimine complex. This complex showed localization on one specific bipyridine by exhibiting dipole direction on linkage with DNA. This complex also showed excitation for an increased amount of time during its linkage with DNA .This complex also undergoes FTIR studies which showed its excited state electrons are well localized on amide-substituted bipyridine. Thus the synthesis of metallo-oligonucleotide were breakthrough for their role in the transfer of energy and electron through complementary oligonucleotide with possible quenchers including the phenothiazine or Os(bpy)32+.

Khan,S.I et al (1999)

The development of triplex forming oligonucleotide(TFO) to target at duplex DNA in order to create specific gene targeting sequence, by the use of antisense oligonucleotide in human or other organism. The base of oligonuctleotide was modified by the incorporation of pyridine stretched analogue in order to increase stabilisation of duplex DNA after base stacking of heterocylic rings. The pyridine stretched base on this occasion used was hypoxanthine by using the solid phase synthesis of oligonuctides method.The synthesized stretched Hypoxanthine Str (H) was then incorporated into different bases of oligonuctleotide such as adenine-stretched hypoxanthine, pyridine-stretched hypoxanthine and benzene-stretched hypoxanthine. The Str (H) base is same with each pair so the increase in stability after observing it through UV thermal melting studies was due to the overlap between adjacent bases responsible in increasing the duplex DNA stability .The Triplex formation of Oligonuctleotide with duplex DNA requires furthermore characterization which is still under research.

Davis, M.L et al (2008)

In 2009, DNA modifications were carried by proposing a new research by designing a series of size expanded adenine (A) by the use of hetero ring expansion technique to observe the effects on change in properties and stability by comparing it with natural counterparts and the previous work done by the same researcher group on the size expanded Guanine analogues. During their work on Guanine analogue it was examined for its structural properties, electronic and spectroscopic properties. The results of this guanine analogue showed prosperous outcomes regarding that artificially synthesized bases incorporated with natural bases can be stable and that the electronic conductivity by these synthesized expanded bases are smaller as compared to natural counterparts. Further examination about the molecular dynamics of this size-expanded base analogue displayed that the helixes of this base analogue is as thermodynamically stable as the Watson & Crick helixes. After these encouraging results the researchers of this group made another design for studying the size-expanded adenine.

The size-expanded adenine research was designed for investigating its base pairing properties through density functional theory (DFT) calculations, molecular dynamics (MD) simulation to determine the stability of duplex in solution. The hetero-ring-expanded base pair for investigating its characteristics after being synthesized was subjected to DFT calculations. A Gaussian 03 set of programs and then the moleculer dynamics simulation were performed .These investigation showed that the base pair are selective while pairing up with natural bases and are stable with their binding energy levels as they remain double stranded. The modified base pairs have good electrical conductivity as compared to natural DNA base pairs and these results of size-expanded bases are useful for biotechnology work of DNA. However the base pair stability and electrical conductivity has increased and is positive for the size-expanded DNA too, as these studies are helpful for further modifications of DNA but there are still different properties which are still remaining to be studied as the DNA protein and DNA ion interactions and their effects of the stability and electrical affinity are still not determined. Han, L et al (2009)

In this research project of size-expanded DNA, the synthesises of adenine nucleoside phosphoramidite base with sugar protected is incorporated in natural DNA ,which will encourage the selectivity ,gene sequencing stability and thermodynamic properties of DNA to function in a genetic system with similar properties as natural DNA base pair. This will be a marked improvement in Triplex forming Oligonucleotides(TFOs) with the duplex DNA and the strands of this modified DNA will work with high affinity and stability when compared with different strategies of DNA modifications. This work will also enhance the chances of using adenine base analogue to be modified and incorporate it within different base pairs like pairing it up with cytosine, thymine and guanine to make them stable on their size expansion for genetic alterations and to target different nucleic acids.

Scheme :Size expanded nucleobases xAdenine of DNA .The box highlights the part where the nucleobases have formally been size-expanded.

Heckel,A. Et al (2004)

If resources are available this research would further be carry out using this Stretched adenine phosphoramidite analogue and will incorporate this with another synthesized Stretched Guanine to form a four base pair DNA. These two pyrimidine analogues would be containing a heterocyclic ring in their structure. It will be a structure like xA-T,T-xA,xG-C,C-xG. This will form a new four base pair genetic system which will encode the genetic information and will function as a natural DNA and if the structure of pyrimidine analogue appears to be as expected then it will be sharing a two and three hydrogen bonds and this is similar to that of natural DNA so it will hopefully be stable. This will form double helixes and these helixes will be tested for their characterization by thermal denaturation, helix stichiometry, Ionic strength dependence, pairing stability, pairing selectivity, mixing of data, Circular Dichoirism Spectra and the characterization of fluorescence activity in DNA base pairs. If the results are successful it would be giving a new dimension for size expanded DNA, as it will be a four base pair analogue which will be highly selective on its sequence of recognition as compared to the natural DNA. The distinct structure of the newly synthesized four base pair DNA will be also be helpful in recognizing the nucleic acid targeting and sequencing.

Figure: Scheme of four base pairing of expanded DNA (xDNA)

Krueger,A.T et al (2007) ; Heckel,A et al (2004)


The aim of the research is to make a size-expanded DNA, by making available the pyridine-stretched adenine nucleoside phosphoramidite in base and sugar protected form for their incorporation into oligomers. The research will involve the solid phase supported automated DNA synthesis and the evaluation of new pyridine-stretched adenine phosphoramidite oligonucleotide.


Benzo-fused design of pyrimidine is formed in order to match the structure of the expanded purine bases beacuase the expanded bases (A, G, C and T) have their own pairing edges. The size-expanded DNA design is conceived in such a way that this analogue of DNA is more likely to be the same as the natural DNA but there are vital differences between them. In expanded DNA, there are eight of the total components of the base pairs. The purine bases which are fused with benzo-ring , are paired with pyrimidines and in the same way the benzo-pyrimidine base is paired with purines having four different rings in between, but in natural DNA there are four components with each of them having purine paired with pyrimidine at their specific positions. This design demonstrates the selectivity of expanded DNA of each component. Krueger, A.T et al (2007)

The research on size-expanded DNA will be carried out in different steps for synthesis. At the start an intermediate will be produced from which the pyridine stretched adenine nucleoside analogue will be synthesized. This intermediate will be yielded by isolating it with a 4-5-nitroimidazole, a crystalline salt to be used after different catalytic reaction. Then after yielding the intermediate which is imidazo [4-5-b] pyridine, it will be further treated with different solvents and after washing at the end with H2O, stretched adenine phosphoramidite analogue will be synthesized.

Phosphoramidite synthesis will be carried out to form adenine stretched analogues by the help of different solvents .This analogue formation is carried out for its incorporation in oligonucleotide. The automated DNA synthesis of oligoneuclotide will be carried out in a DNA synthesizer available for the research along with different reagents which will be required to use during its synthesis in synthesizer. After the synthesis, it will be incubated at 55 °C to make deprotected oligonucleotide. After the deprotection process, the synthesized oligonucleotide will be run through HPLC for its purity testing.

The automated synthesized DNA oligonucleotide would be tested for its stability by analyzing their results of melting profile, known as UV thermal melting studies. This is carried by making the stock solution of separate oligonucleotides and then diluted it into a solution which is being used in work for absorbance. After this the two solutions are combined and the resulting solution is subjected to heat at 90°C for approximately 5 minutes. Then this solution is allowed to cool at room temperature for about 3 hr and then thermal denaturation of the synthesized oligonucleotide is done through spectrophotometer available and then their different temperature variation at different times will be investigated. Clayton, R. et al (2000)

Fluorescence characterization assay will be carried out for the wavelength shifts, enhancements or quenching of emission. The characterization of these properties will help in biophysical analysis and biotechnological use.

In this research the Density functional Theory (DFT) calculations will also be conducted which is about examining the nucleic acid geometries and energies of DNA bases. These are calculations regarding the stable structure having sugar and phosphate as backbone of the stacked base pairs to determine its electronic properties and to estimate its affinity in electrical conductivity in a synthesized DNA. This will help to identify its energy levels during transferring of information in genetic system. Kurita, N. et al (2000)

The Moleculer dynamics (MD) simulation investigation is done for calculating the differences in energy levels of the Stretched Adenine phosphoramidite. This will provide the stability in strands helixes of base incorporated in duplex DNA in a solution. As the solvent have effect on the stability of DNA having the potential energy surfaces (PES) and the free energy surfaces(FES) when the transfer of gene information with hydrogen bonds from one base pair to another takes place.

Maranon,J. et al (1999)

According to possible available resources all these calculations and analysis will be carried out for the characterization and assay for the quantification of the newly synthesized size expanded DNA to function in a genetic system as similar to natural DNA .As these methods are expensive and their equipments and materials are not always present in research labs, then if some of the method's and resources are not available, this design will be altered according to the requirements and will successfully estimate its properties to the best possible resources provided.


Kruguer,A.T,.LU,H.Lee,A.H.F,.Kool,E.T.(2007) Synthesis and Properties of Size-Expanded DNAs:Toward Designed,Functional Genetic Systems,Accounts Of Chemical Research, 40 p:141-150.

This reference is used as it has large diversity of information for Size-expanded DNA about the history,synthesis,benzo-fusion and different important aspects in studies related to genetic system.

Lee,A.H.F,Kool.E.T.(2005) A New Four-Base Genetic Helix ,yDNA,Composed of Widened Benzopyrimidine-Purine Pairs, J.Am.Chem.Soc ,27 p:3332-3338

This reference is used because it has new dimensions in size-expanded DNA with 2 expanded base pairs incorporated in a natural DNA.It has demonstrated studies and results which gives bases for the future research.

Clayton,R.,Davis,M.L.,Fraser,W., Li,W.,Ramsden,C.A.(2002) Synthesis of Pyridine-stretched 2'-Deoxynucleosides,Synlett,9 p:1483-1486

This literature has detail studies about how to synthesize an intermediate and then the streteching of the base. It provides help in understanding the phenomenon involved in synthesizing a size-expanded DNA.

Khan,S.I.,Beilstein,A.E.,Sykora,M.,Smith,G.D.,Hu,X.,Grinstaff,M.W.((1999) Automated Solid-Phase DNA Synthesis and Photophysical Properties of Oligonucleotides Labeled at the 5¢-Terminus with Ru(bpy)3 2+,Inorganic Chemistry ,38 p:3922-39225

From this literature review, the photo-physical studies of a size-expanded DNA base and the phenomenon of solid phase synthesis involved in the development of new stretched DNA base pair is understood.

Han,L., Li,H., Cukier,R.I.,Bu,Y.(2009) Hetero-Ring-Expansion Design for Adenine-Based DNA Motifs: Evidence from DFT Calculations and Molecular Dynamics Simulations.J.Physics and Chemistry ,113


This literature involves the research study on adenine-stretched base and its estimation through DFT Calculations and Moleculer Dynamics simulation has been carried for characterization if the stretched adenine. The research which will be carried out will be on adenine and its stretching by a hetero-cycling ring and will involve these test which are carried in this literature which is used for studying it.

Krueger,A.T., Kool,E.T.(2008) Fluorescence of Size-Expanded DNA Bases: Reporting on

DNA Sequence and Structure with an Unnatural Genetic Set,J.Ame.Chem.Soc ,130, p:3989-3999

To study the process of fluorescence analysis in size-expanded DNA base,its impact on its characterization and its effect on the stability of the base pair .

Mahato,.R.I.,(2007) Pharmaceutical Dosage Forms and Drug Delivery,Biotechnological Pharmaceutical Dosae for .USA,CRC PRESS.

To study the bases of modifying DNA bases and the antisense,Triplex forming oligonucleotides and Stretching of DNA and how to use them for the treatment of genetic disorders.

Heckel,A.,(2004) A new DNA Anlogue with Expanded Size and Scope ,ChemBioChem,5,p:765-767

This literature is used to study the structure of DNA base pairs and their structural difference

with the natural DNA. It also highlights the importance of size expanded DNA to form a new functional genetic system.

Liu,H.,Gao,J.,Kool,E.T.,(2005) Helix-Forming Properties of Size-Expanded DNA,an Alternative Four-Base Genetic Form.J.AM.CHEM.SOC,127,P:1396-1402

It provides the studies which is different for other size expanded DNA base pairs as it in this literature the stretched of DNA takes place in two base pairs of DNA bases and this research provides bases for the future studies in four-base pair modified genetic system by working on different base piars.

Davis,M.L.,Fraser,W.,Ramsden,C.A,(2008) Pyridine-Stretched Oligonucleotide,Synlett.

This literature provides the bases of the research of Stretched Adenine which would be carried out in this research project .The research method design and the characterisation studies have been review from this literature.

Maranon,J.,Fantoni,A.,Grigera,J.R., (1999) Moleculer Dynamics Simulation of Double Proton Transfer:Adenine-Thymine Base Pair , J.Theor.Bio ,201,p:93-102

In order to understand the role of Moleculer Dyanamics simulation in size-expanded DNA and the effects on the selectivity and stability of the DNA.

Machado,M.,Ordejon,P.,Artacho,E.,Portal,D.S.,Soler,S.,(1999) Density Functional Calculations Of Planar DNA base-pairs,J.Phys.Chem.B.p :1-12

To understand the phenomenon of Density Functional Calculations in stretched base pairs incorporated in natural DNA and their impact on the structural and functional system of the DNA. Oligonucleotide.Definition Avaliable from ; [Accessed 28 Feb 2010 ]

This webpage was referred to understand the basic knowledge about oligonuctleotides and antisense system and their role in the development and their role in size expanded DNA.