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Related Concept Videos

Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

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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...
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In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
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In response to DNA damage, cells can pause the cell cycle to assess and repair the breaks. However, the cell must check the DNA at certain critical stages during the cell cycle. If the cell cycle pauses before DNA replication, the cells will contain twice the amount of DNA. On the other hand, if cells arrest after DNA replication but before mitosis, they will contain four times the normal amount of DNA. With a host of specialized proteins at their disposal,cells must use the right protein at...
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Restarting Stalled Replication Forks02:37

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DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
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Translesion DNA Polymerases02:10

Translesion DNA Polymerases

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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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The Replisome03:01

The Replisome

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DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
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Related Experiment Video

Updated: Jan 13, 2026

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System
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Triplex DNA clamp regulates Cas12a activation for ssDNA and RNA sensing.

Andrea Celeste Di Pede1, Neda Bagheri1, Erica Belforte1

  • 1Department of Chemical Science and Technologies, University of Rome, Tor Vergata, Via della Ricerca Scientifica 1, Rome 00133, Italy.

Nucleic Acids Research
|January 8, 2026
PubMed
Summary

We developed a novel CRISPR-Cas12a activation method using triplex DNA formation. This strategy enables sensitive detection of single-stranded DNA (ssDNA) and RNA without specific guide RNAs, improving nucleic acid sensing.

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Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks
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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • CRISPR-Cas12a systems offer precise gene editing and nucleic acid detection capabilities.
  • Current Cas12a assays often require specific guide RNAs and can struggle with single-nucleotide variants or diverse target types.
  • Developing adaptable and highly specific Cas12a detection platforms is crucial for advancing molecular diagnostics.

Purpose of the Study:

  • To engineer a programmable CRISPR-Cas12a activation strategy responsive to triplex DNA formation.
  • To create a Cas12a detection assay that eliminates the need for target-specific guide RNAs.
  • To enhance the specificity and versatility of Cas12a-based nucleic acid sensing.

Main Methods:

  • A molecular strategy was designed to activate CRISPR-Cas12a via triplex DNA formation triggered by single-stranded DNA (ssDNA) or RNA.
  • A triplex-controlled Cas12a assay utilized clamp-like triplex structures and a DNA hairpin (PAM-Switch) for strand displacement.
  • Activation of the Cas12a ribonucleoprotein (RNP) complex initiated trans-cleavage, generating a fluorescent signal.

Main Results:

  • The assay successfully decoupled target recognition from direct hybridization with the Cas12a-crRNA complex, removing the need for target-specific crRNAs.
  • The platform demonstrated enhanced specificity for single-nucleotide variants.
  • Detection of both ssDNA and RNA targets within a 10-20 nucleotide range was achieved using a single Cas12a reaction mix.

Conclusions:

  • The triplex-controlled Cas12a platform offers a programmable and versatile approach for nucleic acid detection.
  • This method overcomes limitations of current Cas12a diagnostics, enabling multiplexed detection and improved specificity.
  • The strategy opens new avenues for developing advanced nucleic acid sensing technologies.