In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Summary

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely applicable for studying protein-nucleic acid interactions.

Abstract

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope ³²P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting ³²P in various nucleic acid-related biochemical assays in vitro. 

Introduction

Protein-nucleic acid interactions are involved in many essential cellular processes such as DNA repair, replication, transcription, RNA processing, and translation. Protein interactions with specific DNA sequences within the chromatin are required for the tight control of gene expression at the transcriptional level¹. Precise posttranscriptional regulation of numerous coding and noncoding RNAs necessitates extensive and complicated interactions between any protein and RNA². These layers of gene expression regulatory mechanism comprise a cascade of dynamic intermolecular events, which are coordinated by interactions of tr....

Protocol

1. Protein preparation

1. MEIOB and MOV10 expression constructs
   1. Generate cDNA expression constructs encoding mouse MEIOB-A, C, and E (Figure 1A) and MOV10.
      1. Set up the polymerase chain reaction (PCR) reactions for each fragment. Mix 1 μL of mouse cDNA (from C57BL/6 mouse testis), 1 μL of dNTP, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 1 μL of DNA polymerase, 25 μL of 2x PCR buffer, an.......

Discussion

Investigating protein-nucleic acid interactions is critical to our understanding of molecular mechanisms underlying diverse biological processes. For example, MEIOB is a testis-specific protein essential for meiosis and fertility in mammals^25,26,27. MEIOB contains an OB domain that binds to single-stranded DNA and exhibits 3' to 5' exonuclease activity²⁶, which directly relates to its ph.......

Materials

Name	Company	Catalog Number	Comments
Equipment
Centrifuge	Eppendorf, Germany	5242R
Chemiluminescent Imaging System	Tanon, China	5200
Digital sonifer	Branson, USA	BBV12081048A	450 Watts; 50/60 HZ
Semi-dry electrophoretic blotter	Hoefer, USA	TE77XP
Tube Revolver	Crystal, USA	3406051
UV-light cross-linker	UVP, USA	CL-1000
Materials
Amicon Ultra-4 Centrifugal Filter	Milipore, USA	UFC801096	4 ml/10 K
Nylon membrane	Thermo Scientific, USA	TG263940A
TC-treated Culture Dish	Corning, USA	430167	100 mm
TC-treated Culture Dish	Corning, USA	430597	150 mm
Microtubes tubes	AXYGEN, USA	MCT-150-C	1.5 mL
Tubes	Corning, USA	430791	15 mL
Reagents
Ampicillin	SunShine Bio, China	8h288h28
Anti-FLAG M2 magnetic beads	Sigma, USA	M8823
ATP	Thermo Scientific, USA	591136
BCIP/NBT Alkaline Phosphatase Color Development Kit	Beyotime, China	C3206
CelLyticTM M Cell Lysis Reagent	Sigma, USA	107M4071V
ClonExpress II one step cloning kit	Vazyme, China	C112
Chemiluminescent Nucleic Acid Detection Kit	Thermo Scientific, USA	T1269950
dNTP	Sigma-Aldrich, USA	DNTP100-1KT
DMEM	Gibco, USA	10569044
DPBS buffer	Gibco, USA	14190-136
EDTA	Invitrogen, USA	AM9260G	0.5 M
EDTA free protease inhibitor cocktail	Roche, USA	04693132001
EndoFree Maxi Plasmid Kit	Vazyme, China	DC202
FastPure Gel DNA Extraction Mini Kit	Vazyme, China	DC301-01
FBS	Gibco, USA	10437028
FLAG peptide	Sigma, USA	F4799
Glycerol	Sigma, USA	SHBK3676
GST Bulk Kit	GE Healthcare, USA	27-4570-01
HEPES buffer	Sigma, USA	SLBZ2837	1 M
IPTG	Thermo Scientific, USA	34060
KoAc	Sangon Biotech, China	127-08-02
Lipofectamin 3000 Transfection Reagent	Thermo Scientific, USA	L3000001
MgCl2	Invitrogen, USA	AM9530G	1 M
NaCl	Invitrogen, USA	AM9759	5 M
NP-40	Amresco, USA	M158-500ML
Opti-MEM medium	Gibco, USA	31985062
PBS	Gibco, USA	10010023	PH 7.4
RNase Inhibitor	Promega, USA	N251B
Streptavidin alkaline phosphatase	Promega, USA	V5591
TBE	Invitrogen, USA	15581044
Tris-HCI Buffer	Invitrogen, USA	15567027	1 M, PH 7.4
Tris-HCI Buffer	Invitrogen, USA	15568025	1 M, PH 8.0
Tween-20	Sangon Biotech, China	A600560