Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry

• Published in: Analytical biochemistry 2004-02, Vol.325 (1), p.137-143
• Main Authors: Rhee, Steven S, Burke, Donald H
• Format: Article
• Language: English
• Subjects: APM–PAGE; ATPγS; Base Sequence; dithiothreitol; Dithiothreitol - chemistry; DTNB; DTT; Enzyme Activation; Kinetics; Molecular Structure; NTB; Phosphates - chemistry; Phosphines - chemistry; Reductant; Ribozyme; RNA; RNA - chemical synthesis; RNA - chemistry; RNA, Catalytic - chemistry; TCEP; Thiophosphorylation; Tris(2-carboxyethyl)phosphine
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2003.10.019

We assessed the utility of the sulfhydryl reductant Tris(2-carboxyethyl)phosphine (TCEP) for both nucleic acid and thiophosphate chemistry, including its effects on organomercurial gel electrophoresis, RNA catalysis, RNA backbone stability, and the intrinsic stability of TCEP. The sulfhydryls of dithiothreitol (DTT) compete with thiophosphates for binding to the mercury within [( N-acryloylamino)phenyl] mercuric chloride (APM) polyacrylamide gels, whereas millimolar concentrations of TCEP gave no difference in the fraction of thiophosphorylated RNA retained on the APM interface relative to samples containing no reductant. Ribozyme activity in TCEP, assessed by the self-thiophosphorylating Kin.46 ribozyme, was unaffected by the presence of DTT or TCEP or by the absence of reductant, as measured on APM gels and evaluated by Michaelis–Menten kinetics. Unexpectedly, TCEP more than doubled the half-life of full-length RNA at 50 and 70 °C, whether in 5 or 50 mM MgCl 2, relative to DTT and the absence of reductant. Under these same conditions, the 5 ′-thiophosphate showed negligible decay, and TCEP was more stable than DTT. TCEP thermostability was equivalent in the presence of 5 or 50 mM MgCl 2 and 10 mM adenosine or ATP.