PMID- 21293790
OWN - NLM
STAT- MEDLINE
DCOM- 20111206
LR  - 20191210
IS  - 1364-5528 (Electronic)
IS  - 0003-2654 (Linking)
VI  - 136
IP  - 17
DP  - 2011 Sep 7
TI  - A simple and rapid approach for measurement of dissociation constants of DNA 
      aptamers against proteins and small molecules via automated microchip 
      electrophoresis.
PG  - 3461-8
LID - 10.1039/c0an00842g [doi]
AB  - Automated microchip electrophoresis was used as a simple and rapid method to 
      measure effective dissociation constants (K(d,eff)) of aptamers against both 
      large and small molecule targets. Human thrombin, immunoglobulin E (IgE), and 
      adenosine triphosphate (ATP) were selected as model analytes to validate the 
      method, with four ligands including two DNA aptamers for thrombin (two distinct 
      epitopes), an IgE aptamer, and an ATP aptamer. The approach is based on a 
      microchip version of a DNA mobility shift assay. Non-denaturing microchip gel 
      electrophoresis separations of DNA could resolve and quantify unbound from 
      target-bound aptamers when using large molecules as targets. To extend the 
      technique to small molecule targets such as ATP, an aptamer/competitor strategy 
      was used, in which a DNA competitor complementary to the aptamer could be 
      displaced by ATP and electrophoretically resolved. Using an automated microchip 
      electrophoresis platform, parallel separations of 11 titration samples were 
      completed in ~0.5 h. Analytical performance comparisons show that our approach 
      provides significant advantages in minimized reagent consumption (typically tens 
      of pmol of aptamer and target), reduced analysis time, and minimized user 
      interaction when compared to previously reported methods for aptamer K(d) 
      measurement. Moreover, the flexibility and ease of K(d,eff) measurement for 
      aptamers against large and small targets make this a unique and valuable approach 
      that should find widespread use. Finally, the feasibility of using this method 
      during aptamer selection processes (e.g. SELEX) was shown by accurate bulk 
      K(d,eff) measurement of a known thrombin aptamer (THRaptA) spiked into a 
      random-sequence DNA pool at as low as 5.0% (molar %) of the total pool; only ~825 
      fmol of total binding sequences were needed for an 11-point titration curve.
FAU - Hu, Jiaming
AU  - Hu J
AD  - Auburn University, Department of Chemistry and Biochemistry, 179 Chemistry 
      Building, Auburn, AL 36849, USA.
FAU - Easley, Christopher J
AU  - Easley CJ
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Validation Study
DEP - 20110203
PL  - England
TA  - Analyst
JT  - The Analyst
JID - 0372652
RN  - 0 (Aptamers, Nucleotide)
RN  - 145563-68-4 (thrombin aptamer)
RN  - 37341-29-0 (Immunoglobulin E)
RN  - 8L70Q75FXE (Adenosine Triphosphate)
RN  - EC 3.4.21.5 (Thrombin)
SB  - IM
MH  - Adenosine Triphosphate/*analysis
MH  - Aptamers, Nucleotide/*chemistry
MH  - Electrophoresis, Microchip/economics/*methods
MH  - Humans
MH  - Immunoglobulin E/*analysis
MH  - SELEX Aptamer Technique
MH  - Sensitivity and Specificity
MH  - Thrombin/*analysis
MH  - Time Factors
EDAT- 2011/02/05 06:00
MHDA- 2011/12/13 00:00
CRDT- 2011/02/05 06:00
PHST- 2011/02/05 06:00 [entrez]
PHST- 2011/02/05 06:00 [pubmed]
PHST- 2011/12/13 00:00 [medline]
AID - 10.1039/c0an00842g [doi]
PST - ppublish
SO  - Analyst. 2011 Sep 7;136(17):3461-8. doi: 10.1039/c0an00842g. Epub 2011 Feb 3.