ABSTRACT
CE application in aptamer selection (CE-SELEX) shows more advantages than other selection methods. In this study, an online reaction based single-step CE (ssCE) mode was employed for fast obtaining protein-ssDNA complex. Using human thrombin (H-Thr) and its aptamer Apt29 as models, we accomplished the procedures of mixing, reaction, separation, detection and complex collection in single step online process, which took about 10â¯min to obtain the H-Thr/Apt29 complex. Important factors, affecting the aptamer and H-Thr interaction (buffer, ratio of aptamer and H-Thr amount), and complex separation and collection (voltage and temperature) were discussed. Later, the online reaction of H-Thr with an 80â¯nt ssDNA library was realized under optimized conditions, and the H-Thr/ssDNA complex was collected and subjected to PCR. By analyzing the PCR product through capillary gel electrophoresis, the resulting approximative 80â¯nt DNA length validated the ssDNA sequence in complex. To confirm the availability of ssCE mode, two ssDNA libraries with different lengths (56â¯nt and 82â¯nt ssDNA) and three proteins (platelet derived growth factor, PDGF-BB; lactoferrin protein, LF; and single-strand DNA binding protein, SSB) were utilized. Their complex peaks were also observed in electropherograms as expected. Additionally, the online incubation of ssDNA and H-Thr was achieved by stopping the separation voltage for some time when ssDNA passed the H-Thr zone. Our results show the ssCE mode has apparent merits of saving time and sample cost for aptamer selection against protein targets.
Subject(s)
DNA, Single-Stranded/chemistry , Electrophoresis, Capillary/methods , Proteins/chemistry , SELEX Aptamer Technique/methods , Aptamers, Nucleotide/chemistry , Becaplermin , DNA-Binding Proteins , Gene Library , Humans , Lactoferrin , Macromolecular Substances/chemistry , Thrombin/chemistryABSTRACT
Peptide Nucleic Acid (PNA) is a nucleic acid analogue, whose neutrally charged and hydrophobic backbone makes it more stable in vivo, so it might act as a potentially better protein probe as compared to aptamer. Currently the investigation of PNA and protein interaction is scarce. In this research, multiple modes of capillary electrophoresis were established and applied for PNA characterization and its interaction with ssDNA and protein. A 15-mer PNA having the same nucleobase sequence as 15-mer anti-thrombin DNA aptamer was chosen as PNA model for this study, its pI (7.71) was estimated by capillary isoelectric focusing (cIEF). Due to its neutral charge and strong hydrophobicity, three micellar electrokinetic chromatography (MEKC) modes containing (a) SDS, (b) Triton X-100 and (c) CTAB were compared for PNA related analysis. CTAB was not applicable for PNA analysis, while in 4mM SDS or 2mM Triton X-100, PNA and PNA-ssDNA complex can be identified directly. The significant peak of PNA-ssDNA complex helped in validating the two MEKC modes for PNA and target interaction study. Furthermore, the effect of SDS and Triton X-100 concentrations in the two MEKC modes on the protein target thrombin analysis was investigated by capillary zone electrophoresis (CZE). 4mM SDS caused thrombin denaturation. So in 2mM Triton X-100, interactions of PNA with thrombin, PNA with RNase A and a non-aptameric PNA (n-PNA) with thrombin were compared. PNA with thrombin exhibited strongest binding. In summary, cIEF mode for PNA pI determination, MECK mode for direct PNA, PNA-ssDNA and PNA-protein complex identification and CZE mode for the effect of surfactant in MEKC modes on protein target thrombin analysis were applied. Above results showed that multiple modes of CE provide rapid and very low-sample cost methods for PNA related studies.
Subject(s)
Electrophoresis, Capillary/methods , Peptide Nucleic Acids/chemistry , Thrombin/chemistry , Micelles , Octoxynol/chemistry , Protein Binding , Surface-Active Agents/chemistryABSTRACT
This paper reviews the capillary electrophoresis (CE) in 2014. Five international and two national conferences are included and the important reports are introduced briefly. The literatures from ISI Web of Science published in 2014 (Jan. 1st to Dec. 15th) are classified and introduced based on the biology and medicine applications, the use of detectors as well as the important analytical chemical journals. In the end, CE developments in 2012-2014 are reviewed and compared.
