Mixed-Sequence Recognition of Double-Stranded DNA Using Enzymatically Stable Phosphorothioate Invader Probes.
Brooke A Anderson1, Saswata Karmakar2, Patrick J Hrdlicka3
1Department of Chemistry, University of Idaho, Moscow, ID 83844-2343, USA. brookea@scripps.edu.
Molecules (Basel, Switzerland)
|August 1, 2015
Summary
New phosphorothioate DNA Invader probes offer enhanced stability for targeting double-stranded DNA (dsDNA). These sequence-unrestricted probes show promise for gene regulation and manipulation in cellular environments.
Area of Science:
- Molecular Biology
- Biochemistry
- Chemical Biology
Background:
- Developing sequence-unrestricted double-stranded DNA (dsDNA) recognition probes is crucial for gene-targeting applications.
- Established methods like triplex-forming oligonucleotides, peptide nucleic acids, and polyamides have limitations.
- Invader probes, utilizing intercalator-functionalized nucleotides, offer an alternative approach to dsDNA recognition.
Purpose of the Study:
- To characterize Invader probes with phosphorothioate backbones (PS-DNA Invaders).
- To evaluate the stability and dsDNA recognition efficiency of PS-DNA Invaders.
- To assess the potential of PS-DNA Invaders for applications in cellular environments.
Main Methods:
- Synthesis of Invader probes with phosphorothioate backbones.
- Characterization of probe stability against nucleolytic degradation.
- Assessment of dsDNA recognition efficiency using biophysical techniques.
Main Results:
- PS-DNA Invader probes exhibit significantly enhanced stability against nucleolytic degradation compared to phosphodiester-based probes.
- These probes maintain acceptable efficiency in recognizing and binding to dsDNA targets.
- The modified backbone does not impede the fundamental mechanism of Invader probe activation and dsDNA interaction.
Conclusions:
- PS-DNA Invader probes represent a promising advancement for dsDNA-targeting applications.
- Their increased stability makes them suitable for use in complex biological systems.
- Further development could lead to novel tools for gene detection, regulation, and manipulation.
More Related Videos
Related Concept Videos
01:05Maxam-Gilbert Sequencing
13.7K
In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The...
Challenges of the Maxam-Gilbert Method
The...
13.7K
03:00Next-generation Sequencing
101.7K
The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features....
101.7K
03:31Labeling DNA Probes
9.8K
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
9.8K
01:03Single-Strand DNA Binding Proteins
17.2K
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...
17.2K
02:57Southern Blot
24.6K
Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
24.6K
01:57Sanger Sequencing
779.2K
DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
779.2K