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Sequence Modifications
 

Modifications of oligonucleotides can be very versatile. Here, in addition to terminal modifications of the 3´-or 5´-end of an oligonucleotide, also internal changes in the sequence are possible. The incorporation of certain nucleosides into the base sequence can sometimes also considerably affect the behavior of cells in the organism. 
Below we present some of our sequence modifications of oligonucleotides available with us.

Wobbles

Wobbles


In molecular genetics, the wobble hypothesis explains how a particular tRNA can pair more than one mRNA triplet (codon). The first two positions of the codon usually bind to complementary bases of the anticodon of tRNA. Only the first position of the anticodon is not always specified and due to utilisation of rare bases like inosine, unusual base pairings are possible (wobble position).

Possible variants:

Guanosine : Uridine Inosine : Uridine Inosine : Adenosine Inosine : Cytidine
wobble base pairing
 

Oligonucleotides with wobbles (mixed bases, degenerate bases) can be used in many applications. During the synthesis of oligonucleotides wobbles can be incorporated at defined positions. Depending on the type of mixing base, specific statistical distributions of bases can be generated at each position in the oligo. 

Synthesis of oligos with wobbles: 
Many modern DNA/RNA synthesizer “understand” the one letter code for wobbles (see below) and are able to mix the respective building blocks in the right proportion during the synthesis (2-wobble: 50% of each component,
3-wobble: 33% each and N=4-wobble: 25%). This quick and easy method usually leads to good results with small deviations from the desired statistical distribution. Depending on the particular sequence and the difference in reactivity of the building blocks, in individual cases the deviations can be higher. 
In critical cases or larger synthetic approaches manual mixing of the bases in the right proportion before the synthesis of the oligos can significantly improve the results. Furthermore, mixtures are also possible that deviate from a uniform distribution. For further information please contact our customer support team.

Purification of oligos with wobbles:
Depending on the type and number of wobbles the synthesised oligonucleotide is composed of a variety of different sequences. These, possibly several thousand sequences may differ for example in their mobility in polyacrylamide gels (PAGE) or on the HPLC column. Oligos having many wobbles in their sequence lead to a broadening of the corresponding gel band or peak in the HPLC. Especially when cutting bands out of gel there is a risk that single sequences of the wobble mix get "lost". According to this we do not recommend PAGE purification of oligonucleotides with mixed bases, or only after consultation with our technical support team. 
The following internationally valid "one letter code" shows  the possible wobble combinations of the four standard bases. Please use this code when ordering oligonucleotids with degenerate bases.
 

  Symbol   Explanation   Bases                      
  Normal bases        
A Adenine A      
C Cytosine   C    
G Guanine     G  
T Thymine       T
U Uracil (only RNA)       U
  Wobble bases        
W Weak (binding bases) A     T
S Strong (binding bases)   C G  
M aMino A C    
K Keto     G T
R puRine A   G  
Y pYrimidine   C   T
B Not A (B comes after A)   C G T
D Not C (D comes after C) A   G T
H Not G (H comes after G) A C   T
V Not T (V comes after T and U) A C G  
N aNy base A C G T
 

Deoxyinosine (dI)

Deoxyinosine (dI)

 

In addition to the four DNA bases, adenine, cytosine, guanine and thymine, the bases uracil and inosine and other rare nucleotides can be found in RNA. Inosine is an extremely rare nucleoside of RNA and it is composed of the sugar β-D-ribofuranose and the base hypoxanthine.

Despite the fact that inosine is a purine, inosine may enter into pairings with pyrimidines as well as purines of DNA. According to this, inosine is also known as "universal" or "neutral" base. However, some base pairings seem to be energetically more favorable than others, so that the strength of the bonds between the individual bases differs significantly.

The strength of the bond decreases over the following series:1,2 
I:C > I:A > I:G ≥ I:T

The affinity of inosine to the four other bases is also highly influenced by the surrounding sequence. Despite the structural analogy to guanosine, inosine shows a weaker binding than a guanosine in the same surrounding sequence. Furthermore, the two purines need more space, thus pairings of the bases I:A and I:G also lead to an altered DNA topology.

Particularly, in tRNAs, inosine has biological relevance. The first base of the anticodon (= third position of the codon of the mRNA) is often inosine, so that this position can be flexible assigned. These so-called "wobble" bases allow limited mismatches in the third position of codons.






Desoxyinosine dI
The following table shows the possible mismatches of bases:
tRNA A C G U I
mRNA U G C A A
      U G C
          U
          G


This wobble effect is also possible in oligonucleotides. Due to integration of an inosine nucleotide to the sequence of a primer, the primers obtain "variable" bases. These degenerate primers allow e.g. annealing and amplification of multiple related sequences. While using degenerate primers in PCR, DNA polymerases, such as Pfu, which have a so-called proof-reading function, have been reported to fall off the DNA as soon as they meet wobble bases in the sequence of the primer.3

Order information:
Please be aware to enter deoxy-inosine, as well as ribo-inosine (2´-dInosine, rI) as internal modifications in the order. Identify the position of the internal modification in your sequence by a number (5,6,7,8), which mark the corresponding pull-down menu in the online order form. If no modifications are available for selection, neither the scale “XS” nor the purification “cartridge” must be selected for ribobases.
 



Literature: 
1. Studies on the base pairing properties of deoxyinosine by solid phase hybridisation to oligonucleotides. Case-Green SC, Southern EM; Nucleic Acids Res. (1994), 22(2):131-6.

2. Base pairing involving deoxyinosine: implications for probe design. Martin FH, Castro MM, Aboul-ela F, Tinoco I Jr.;  Nucleic Acids Res. (1985), 13: 8927-8938.

3. PCR with degenerate primers containing deoxyinosine fails with Pfu DNA Polymerase. Knittel T, Picard D, PCR Methods and Applications (1993), 2: 346-347.

2´-deoxy-Uridine (dU)

Deoxyuridine (dU)

 

The nucleoside deoxyuridine (dU) consists of the sugar β-D-deoxyribose and the nucleobase uracil.
Deoxyuridine is structurally very similar to deoxythymidine (dT). Merely by the absence of the 5´-methyl group in deoxyuridine the two nucleosides can be distinguished. The methyl group of deoxythymidine has a slight stabilising effect on the double stranded form of DNA.

Occasionally, deoxyuridine occurs in living cells by hydrolytic deamination of cytidine. The initiation of a repair program in the cell can prevent false pairing during replication of DNA. 
Using the uracil-DNA glycosylase, uracils can be specifically removed from the DNA, creating abasic sites, which can be detected by endonucleases. In this way, strand breaks of DNA can be produced selectively. 
This process is used to specifically remove primers from the reaction mix, e.g. for direct sequencing of PCR products. After a successful PCR reaction, the remaining primer can be used directly for sequencing of the PCR product.

Order information:
Please enter deoxybases, as well as ribobases and other modifications (5-hm-dU, 5-Br-U, 5-Br-dU, 2´-F-dU, 2´-OMe-U) as internal modifications in the online order form. Substitute in the sequence field the position of the internal modification in your sequence by a number (5-8), which mark the corresponding pull-down menu in the online order form. Choose the desired modification from the pull-down menu to specify the symbolising number (5-8).







2ยด-deoxyuridine dU