Head-to-head comparison of LNA, MPγPNA, INA and Invader probes targeting mixed-sequence double-stranded DNA

2020 ◽  
Vol 18 (1) ◽  
pp. 56-65 ◽  
Author(s):  
Raymond G. Emehiser ◽  
Eric Hall ◽  
Dale C. Guenther ◽  
Saswata Karmakar ◽  
Patrick J. Hrdlicka
Keyword(s):  
Specific Recognition ◽  
Double Stranded Dna ◽  
Mixed Sequence ◽  
Modified Dna ◽  
Dna Strands

Double-stranded (ds) Invader and INA probes allow for efficient and specific recognition of mixed-sequence dsDNA targets, whereas recognition is less efficient and specific with single-stranded LNA-modified DNA strands and fully modified MPγPNAs.

scholarly journals Impact of non-nucleotidic bulges on recognition of mixed-sequence dsDNA by pyrene-functionalized Invader probes

2020 ◽  
Vol 18 (24) ◽  
pp. 4645-4655
Author(s):  
Dale C. Guenther ◽  
Raymond G. Emehiser ◽  
Allison Inskeep ◽  
Saswata Karmakar ◽  
Patrick J. Hrdlicka
Keyword(s):  
Specific Recognition ◽  
Double Stranded Dna ◽  
Mixed Sequence

Invader probes featuring non-nucleotidic bulges are energetically activated for highly specific recognition of complementary double-stranded DNA targets.

A colorimetric method for the sequence-specific recognition of double-stranded DNA on the surface of a silver-coated glass slide

2018 ◽  
Vol 96 (5) ◽  
pp. 466-470
Author(s):  
Xiaoting Guo ◽  
Jing Wang ◽  
Zhifang Zhu ◽  
Manjun Zhang ◽  
Haigang Li ◽  
...  
Keyword(s):  
Colorimetric Method ◽  
Colour Change ◽  
Glass Slide ◽  
Coated Glass ◽  
Specific Recognition ◽  
Double Stranded Dna ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Good Sequence ◽  
Coated Glass Slide

In this study, a colorimetric method for sequence-specific recognition of double-stranded DNA (dsDNA) was established on the surface of a silver-coated glass slide. Oligo-1 was assembled on the surface of a silver-coated glass slide through an Ag–S bond, and Oligo-2 as reporter was used to bind with streptavidin-horseradish peroxidase (SA–HRP). They could bind with target dsDNA that was composed of Oligo-3 and Oligo-4 on the surface of a silver-coated glass slide through triplex formation. The bound HRP could be moved into the solution by DNase I and catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). Therefore, the concentration of target dsDNA could be determined with the colour change of TMB. Under the optimum conditions, the absorbance was proportional to the concentration of target dsDNA over the range of 100 pmol/L to 2.0 nmol/L, with a detection limit of 13 pmol/L. In addition, this method showed good sequence selectivity, enabling it to be further developed for the detection of other polymerase chain reaction (PCR) products.

Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination

2003 ◽  
Vol 36 (4) ◽  
pp. 429-453 ◽  
Author(s):  
Chantal Prévost ◽  
Masayuki Takahashi
Keyword(s):  
Homologous Recombination ◽  
Structural Information ◽  
Double Helix ◽  
Genetic Material ◽  
Strand Exchange ◽  
Double Stranded Dna ◽  
Dna Deformation ◽  
Architectural Proteins ◽  
Dna Strands ◽  
Dna Strand

1. Introduction 4302. Transformations of the RecA filament 4312.1 The different forms of the RecA filament 4312.2 Orientation and position of the RecA monomers in the active filament 4332.3 Transmission of structural information along the filament 4333. RecA-induced DNA deformations 4353.1 Characteristics of RecA-bound DNA 4353.2 Stretching properties of double-stranded DNA 4363.3 DNA bound to architectural proteins 4373.4 Implications for RecA-induced DNA deformations 4383.5 Axial distribution of the DNA stretching deformation 4384. Contacts between RecA and the DNA strands 4404.1 The DNA-binding sites 4404.2 Possible arrangement of loops L1 and L2 and the three bound strands of DNA 4425. Strand arrangement during pairing reorganization 4445.1 Hypotheses for DNA strand association 4445.2 Association via major or minor grooves 4465.3 Post-strand exchange geometries 4466. Conclusion 4477. Acknowledgments 4488. References 448Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.

Selective recognition of G-quadruplexes by a dimeric carbocyanine dye

RSC Advances ◽  
2016 ◽  
Vol 6 (90) ◽  
pp. 87400-87404 ◽  
Author(s):  
P. Chilka ◽  
P. R. Patlolla ◽  
B. Datta
Keyword(s):  
Selective Recognition ◽  
Dna Molecules ◽  
Double Stranded Dna ◽  
Mixed Sequence ◽  
Carbocyanine Dye ◽  
G Quadruplex

A novel dimeric carbocyanine dye is found to recognise G-quadruplex structures selectively compared to mixed sequence or double-stranded DNA molecules.

scholarly journals Phosphorothioate-modified DNA oligonucleotides inactivate CRISPR-Cpf1 mediated genome editing

2018 ◽  
Author(s):  
Bin Li ◽  
Chunxi Zeng ◽  
Wenqing Li ◽  
Xinfu Zhang ◽  
Xiao Luo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Adaptive Immune System ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Dna Oligonucleotides ◽  
Double Stranded Dna ◽  
Modified Dna ◽  
Target Dna ◽  
Editing Activity ◽  
Dose Dependent

CRISPR-Cpf1, a microbial adaptive immune system discovered from Prevotella and Francisella 1, employs a single-stranded CRISPR RNA (crRNA) to induce double stranded DNA breaks1. To modulate genome editing activity of Cpf1 in human cells, we designed a series of crRNA variants including DNA-crRNA and RNA-crRNA duplexes, and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1 mediated gene editing. More importantly, without prehybridization, this PS-modified DNA was able to regulate Cpf1 activity in a time-and dose-dependent manner. Mechanistic studies indicate that PS-modified DNA oligonucleotides hinder the binding between Cpf1-crRNA complex and target DNA substrate. Consequently, phosphorothioate-modified DNA oligonucleotides provide a tunable platform to inactivate Cpf1 mediated genome editing.

scholarly journals On deterministic 1-limited 5′ → 3′ sensing Watson–Crick finite-state transducers

2021 ◽  
Vol 55 ◽  
pp. 5
Author(s):  
Benedek Nagy ◽  
Zita Kovács
Keyword(s):  
Dna Computing ◽  
Finite Automata ◽  
Theoretical Computer Science ◽  
Theoretical Computer ◽  
Double Stranded Dna ◽  
Finite State Transducers ◽  
Special Cases ◽  
Finite State ◽  
Dna Strands ◽  
Processing Order

Finite automata and finite state transducers belong to the bases of (theoretical) computer science with many applications. On the other hand, DNA computing and related bio-inspired paradigms are relatively new fields of computing. Watson–Crick automata are in the intersection of the above fields. These finite automata have two reading heads as they read the upper and lower strands of the input DNA molecule, respectively. In 5′ → 3′ Watson–Crick automata the two reading heads move in the same biochemical direction, that is, from the 5′ end of the strand to the direction of the 3′ end. However, in the double-stranded DNA, the DNA strands are directed in opposite way to each other, therefore 5′ → 3′ Watson–Crick automata read the input from the two extremes. In sensing 5′ → 3′ automata the automata sense if the two heads are at the same position, moreover, the computing process is finished at that time. Based on this class of automata, we define WK transducers such that, at each transition, exactly one input letter is being processed, and exactly one output letter is written on a normal output tape. Some special cases are defined and analyzed, e.g., when only one of the reading heads is being used and when the transducer has only one state. We also show that the minimal transducer is uniquely defined if the transducer is deterministic and it has marked output, i.e., the output letter written in a step identifies the reading head that is used in that transition. We have also used the functions ‘processing order’ and ‘reading heads’ to analyze these transducers.

scholarly journals CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

2020 ◽  
Author(s):  
Joshua C. Cofsky ◽  
Deepti Karandur ◽  
Carolyn J. Huang ◽  
Isaac P. Witte ◽  
John Kuriyan ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Strand Break ◽  
Loop Structure ◽  
Loop Formation ◽  
Strand Breaks ◽  
Guide Rna ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
Type V

ABSTRACTMost type V CRISPR-Cas interference proteins use a single RuvC active site to make RNA-guided breaks in double-stranded DNA substrates, an activity essential for both bacterial immunity and genome editing applications. The best-studied of these enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the guide RNA displaces one of the DNA strands of a double-helical substrate, positioning the DNase active site for first-strand cleavage. However, crystal structures and biochemical data have not explained how the second strand is cut to complete the double-strand break. Here, we show that Cas12a-mediated R-loop formation destabilizes DNA at the second-strand cleavage site, which is located outside of the R-loop structure and beyond the 3′ end of the guide RNA. Chemical and fluorescent DNA probes reveal that this destabilization is an intrinsic feature of DNA flanking the RNA-3′ side of R-loops and does not require direct protein interactions. Interestingly, DNA flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-loop structure may explain the uniformity of guide RNA architecture and the single-active-site cleavage mechanism that are fundamental features of all type V CRISPR-Cas systems.

scholarly journals Mechanistic Insights into theCis-andTrans-acting Deoxyribonuclease Activities of Cas12a

2018 ◽  
Author(s):  
Daan C. Swarts ◽  
Martin Jinek
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Cleavage Product ◽  
List Type ◽  
Francisella Novicida ◽  
Ssdna Binding ◽  
Double Stranded Dna ◽  
Target Dna ◽  
Dna Strands ◽  
Dna Strand

HIGHLIGHTSTarget ssDNA binding allosterically induces unblocking of the RuvC active sitePAM binding facilitates unwinding of dsDNA targetsNon-target DNA strand cleavage is prerequisite for target DNA strand cleavageAfter DNA cleavage, Cas12a releases the PAM-distal DNA productSUMMARYCRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA incisand non-target single stranded DNAs (ssDNA) intrans.To elucidate the molecular basis for both deoxyribonuclease cleavage modes, we performed structural and biochemical studies onFrancisella novicidaCas12a. We show how crRNA-target DNA strand hybridization conformationally activates Cas12a, triggering itstrans-acting, non-specific, single-stranded deoxyribonuclease activity. In turn,cis-cleavage of double-stranded DNA targets is a result of PAM-dependent DNA duplex unwinding and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent,trans-active state. Together, these results provide a revised model for the molecular mechanism of Cas12a enzymes that explains theircis- andtrans-acting deoxyribonuclease activities, and additionally contribute to improving Cas12a-based genome editing.

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