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Nucleic Acid Structure

Nucleic Acid Structure

The pentose sugar in DNA is deoxyribose, while in RNA the pentose sugar is ribose. The difference between the sugars is the presence of the hydroxyl group on the ribose's second carbon and a hydrogen on the deoxyribose's second carbon. The phosphate residue attaches to the hydroxyl group of the 5′ carbon of one sugar and the hydroxyl group of the 3′ carbon of the sugar of the next nucleotide, which forms a 5′ to 3′ phosphodiester linkage.
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Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...

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For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...

Nucleic Acids

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Nucleic acids are the most important macromolecules for the continuity of life. They carry the cell's genetic blueprint and carry instructions for its functioning.
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Nucleic acids

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Nucleic acids are the most important macromolecules for the continuity of life. They carry the cell's genetic blueprint and carry instructions for its functioning.
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RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
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Updated: Nov 7, 2025

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

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Published on: May 24, 2017

Mapping RNA with Nuclease S1.

Michael R Green, Joseph Sambrook

Cold Spring Harbor Protocols

|May 4, 2021

Summary

This protocol details nuclease S1 mapping of messenger RNA (mRNA) using a uniformly labeled DNA probe. The method involves creating DNA-RNA hybrids, digesting them with nuclease S1, and analyzing products via gel electrophoresis and radiography.

Area of Science:

Molecular Biology
Biochemistry
Genetics

Background:

Accurate quantification and characterization of messenger RNA (mRNA) are crucial for understanding gene expression.
Nuclease S1 mapping is a widely used technique for analyzing RNA structure and abundance.
Existing protocols require optimization for clarity and reproducibility.

Purpose of the Study:

To provide a detailed protocol for nuclease S1 mapping of mRNA.
To outline the use of uniformly labeled single-stranded DNA probes for enhanced sensitivity.
To ensure clear methodology for DNA-RNA hybrid formation, digestion, and analysis.

Main Methods:

Utilizing uniformly labeled, single-stranded DNA probes for hybridization with target mRNA.
Formation of DNA-RNA hybrids under specific annealing conditions.

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DNAzyme-dependent Analysis of rRNA 2’-O-Methylation

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Enzymatic digestion of DNA-RNA hybrids using nuclease S1 to remove single-stranded DNA or RNA.

Separation of protected DNA fragments by gel electrophoresis.

Detection and analysis of DNA fragments using radiography.

Main Results:

Successful generation of DNA-RNA hybrids suitable for nuclease S1 digestion.
Demonstration of nuclease S1's efficacy in selectively degrading unprotected nucleic acid strands.
Clear resolution of protected DNA fragments corresponding to specific mRNA transcripts via gel electrophoresis.
Accurate mapping and relative quantification of mRNA species through radiographic analysis.

Conclusions:

The described protocol provides a robust and reproducible method for nuclease S1 mapping of mRNA.
This technique allows for precise determination of transcription start sites and mRNA abundance.
The protocol is valuable for researchers studying gene expression regulation and RNA biology.