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.
Subject(s)
Oligonucleotides/chemistry , DNA Ligase ATP , DNA Ligases/metabolism , Imidazoles/chemistry , Oligonucleotides/chemical synthesisABSTRACT
A novel detection method for RNA pathogens based on one-step reverse transcription PCR is introduced here. This method utilized the reverse transcriptase activity and the 5'-nuclease activity of TaqM1 DNA polymerase to transform target RNA into cDNA. The following PCR process released a fragment from the 5' end as a specific probe. Afterwards this fragment triggered a strand-displacement based signal amplification to release large amounts of G-quadruplex DNAzymes. All the probes applied in our method were unmodified DNA oligonucleotides. The detection results could be reported without sophisticated instruments either in the colorimetric way through oxidizing ABTS or in the fluorometric way by using tyramine as substrate. This approach could successfully detect HIV-1 in a blood sample and it has a linear concentration range of 6 fM to 60 pM.
Subject(s)
HIV Infections/diagnosis , HIV-1/isolation & purification , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction/methods , DNA, Complementary/analysis , DNA, Complementary/genetics , Fluorometry/methods , HIV Infections/blood , HIV-1/genetics , Humans , RNA, Viral/blood , RNA, Viral/genetics , RNA, Viral/metabolism , Sensitivity and Specificity , Taq Polymerase/metabolismABSTRACT
A metal-free artificial restriction DNA cutter which is composed of cyclen and classical peptide nucleic acid (PNA) was synthesized. Analysis of DNA cleavage products indicates the site-selective hydrolysis.
Subject(s)
DNA/chemistry , Heterocyclic Compounds/chemistry , Peptide Nucleic Acids/chemistry , Binding Sites , Cyclams , DNA Cleavage , Hydrolysis , Models, Molecular , Nucleic Acid Conformation , Substrate SpecificityABSTRACT
Finding the right fit: Herein, we report on the development of novel steric probes and present initial insights into their interplay with DNA polymerases. Our findings provide experimental evidence for varied enzyme-substrate interactions that might account for the varied selectivity previously observed.
