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. 2014 Apr 4:4:4595.
doi: 10.1038/srep04595.

Template-directed chemical ligation to obtain 3'-3' and 5'-5' phosphodiester DNA linkages

Haodong Chen  1 , Feng Du  2 , Gangyi Chen  2 , Frank Streckenbach  2 , Afshan Yasmeen  2 , Yun Zhao  3 , Zhuo Tang  1
Affiliations

Template-directed chemical ligation to obtain 3'-3' and 5'-5' phosphodiester DNA linkages

Haodong Chen et al. Sci Rep. .

Abstract

Up to now, the direct ligation of two DNA fragments with opposite directions to obtain 3'-3' or 5'-5' phosphate ester bonds is still challenging. The only way to obtain DNA oligonucleotides containing a 3'-3' or 5'-5' inversion of polarity sites is based on professional DNA chemical synthesis. Herein, we demonstrate a convenient template-directed chemical ligation that enables 3'-3' and 5'-5' linkages of two DNA oligonucleotides. This method is based on the assembly of two oligonucleotides on a template in opposite directions through forming antiparallel and parallel duplexes simultaneously, followed by coupling with N-Cyanoimidazole under mild condition. Moreover, on the basis of DNA oligonucleotides with 5'-5' linkage obtained through our template-directed chemical ligation, we developed a new cDNA display technique for in vitro selection of functional polypeptides.

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Figures

Figure 1
Figure 1. Ligation reactions catalyzed by different mechanisms.
(A) Normal 5′-3′ ligation catalyzed by DNA ligase. At the presence of template, ATP and T4 DNA ligase, two oligonucleotides were ligated together and a 5′-3′ phosphodiester bonds was formed. (B) Template-directed chemical ligation of 3′-3′ and 5′-5′ oligonucleotides activated by the coupling reagent N-Cyanoimidazole. Arrows in red represent the parallel oligonucleotide with template.
Figure 2
Figure 2. Time-dependent 3′-3′ chemical ligation.
(A) Proposed mechanism for the chemical ligation promoted by N-Cyanoimidazole in the presence of template and Zn2+ when incubated at 20°C. (B) PAGE analysis of 3′-3′ ligation with different reaction time. Lane 1-7: Ligation reaction for 0, 3, 6, 9, 12, 24 and 48 h. Graph was determined by phosphor imager calculation of gel. 32P in red denotes a labeled phosphate. Full-length blot images are available in Supplementary Figure S10.
Figure 3
Figure 3. Ligation reactions by using N-Cyanoimidazole.
(A) Lane 1: Marker of 5′-P32 labeled P-b; Lane 2: 3′-3′ ligation without N-Cyanoimidazole; Lane 3: 5′-3′ ligation that is similar as in 3′-3′ ligation in length. P-a' has the same sequence with that of P-a in opposite direction; Lane 4: 3′-3′ ligation under standard reaction condition; Lane 5: 5′-3′ ligation which similar as in 5′-5′ ligation in length; Lane 6: 5′-5′ ligation under standard reaction condition; Lane 7: same as lane 6, 5′-5′ ligation by using P-b1 that has one more A than P-b at 5-end. Lane 8: marker of 5′-P32 labeled Template-33. (B) Verification experiments of 3′-3′ chemical ligation. Lane 1: Ligation reaction containing 5′-P32 labeled P-a ligated to 3′-end phosphorylated oligonucleotides P-b in the presence of Template-33; Lane 2: the same reaction as lane 1 but without P-b; Lane 3: the same reaction as lane 1 but without Template-33; Lane 4: Marker of 5′-P32 labeled P-a; Lane 5: Ligation reaction containing 5′-P32 P-b with 3′-phosphorylated oligonucleotides P-a in the presence of Template-33; Lane 6: the same reaction as lane 5 but without P-a; Lane 7: the same reaction as lane 5 but without Template-33; Lane 8: Marker of 5′-P32 labeled P-b; Lane 9: marker. P32 in red indicated the isotope labeling position. The asterisk denotes isotope labeling. Full-length blot images are available in Supplementary Figure S10.
Figure 4
Figure 4. The application of oligonucleotides with 5′-5′ linkage in DNA display.
(A) mRNA-peptide fusion obtained from mRNA diplay. (B) The mechanism of DNA display strategy: 55-Primer containing 5′-5′ linkage with hybridized with mRNA that encode a polypeptide contains 16 amino acids (16aa). During the in vitro translation of mRNA, the puromycin will enter the A site of ribosome to capture the nascent peptide when the ribosome comes to the junction of single stranded RNA and the duplex region. After reverse transcription, the cDNA-peptide fusion is obtained. P in yellow represents puromycin. (C) Result of in vitro translation. RNA Template and puromycin-tethered, 5′-5′ structure-included DNA sequence 55-primer. Lane 1: In vitro translation mRNA in presence of P32 labeled 55-primer. The upper band represents the DNA-peptide fusion (16aa Fusion); Lane 2: In vitro translation containing P32 labeled 55-primer without RNA template as negative control, (D) Reverse transcription of 16aa Fusion on mRNA template. Lane 1: Reverse transcription containing P32 labeled 16aa Fusion and mRNA. The upper band represents the reverse transcription product cDNA-peptide fusion; Lane 2: reverse transcription without RNA template as negative control. Full-length blot images are available in Supplementary Figure S10.
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