Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 8 de 8
Filter
Add more filters










Database
Language
Publication year range
1.
Chemistry ; 27(56): 13991-13997, 2021 Oct 07.
Article in English | MEDLINE | ID: mdl-34382264

ABSTRACT

The series of unnatural base pairs (UBPs) developed by the Romesberg lab, which pair via hydrophobic and packing interactions have been replicated, transcribed, and translated inside of a living organism. However, as to why these UBPs exhibit variable fidelity and efficiency when used in different contexts is not clear. In an effort to gain some insights, we investigated the thermal stability and pairing selectivity of the (d)NaM-(d)TPT3 UBP in 11nt duplexes via UV spectroscopy and the effects on helical structure via CD spectroscopy. We observed that while the duplexes containing a UBP are less stable than fully natural duplexes, they are generally more stable than duplexes containing natural mispairs. This work provides the first insights connecting the thermal stability of the (d)NaM-(d)TPT3 UBP to the molecular mechanisms for varying replication fidelity in different sequence contexts in DNA, asymmetrical transcription fidelity, and codon:anticodon interactions and can assist in future UBP development.


Subject(s)
Base Pairing
2.
Nat Chem Biol ; 17(8): 906-914, 2021 08.
Article in English | MEDLINE | ID: mdl-34140682

ABSTRACT

The development of unnatural base pairs (UBPs) has greatly increased the information storage capacity of DNA, allowing for transcription of unnatural RNA by the heterologously expressed T7 RNA polymerase (RNAP) in Escherichia coli. However, little is known about how UBPs are transcribed by cellular RNA polymerases. Here, we investigated how synthetic unnatural nucleotides, NaM and TPT3, are recognized by eukaryotic RNA polymerase II (Pol II) and found that Pol II is able to selectively recognize UBPs with high fidelity when dTPT3 is in the template strand and rNaMTP acts as the nucleotide substrate. Our structural analysis and molecular dynamics simulation provide structural insights into transcriptional processing of UBPs in a stepwise manner. Intriguingly, we identified a novel 3'-RNA binding site after rNaM addition, termed the swing state. These results may pave the way for future studies in the design of transcription and translation strategies in higher organisms with expanded genetic codes.


Subject(s)
Eukaryota/enzymology , RNA Polymerase II/genetics , Transcription, Genetic/genetics , Base Pairing , Molecular Dynamics Simulation , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism
3.
Nat Chem Biol ; 16(5): 570-576, 2020 05.
Article in English | MEDLINE | ID: mdl-32251411

ABSTRACT

Natural organisms use a four-letter genetic alphabet that makes available 64 triplet codons, of which 61 are sense codons used to encode proteins with the 20 canonical amino acids. We have shown that the unnatural nucleotides dNaM and dTPT3 can pair to form an unnatural base pair (UBP) and allow for the creation of semisynthetic organisms (SSOs) with additional sense codons. Here, we report a systematic analysis of the unnatural codons. We identify nine unnatural codons that can produce unnatural protein with nearly complete incorporation of an encoded noncanonical amino acid (ncAA). We also show that at least three of the codons are orthogonal and can be simultaneously decoded in the SSO, affording the first 67-codon organism. The ability to incorporate multiple, different ncAAs site specifically into a protein should now allow the development of proteins with novel activities, and possibly even SSOs with new forms and functions.


Subject(s)
Base Pairing , Codon , Genetic Engineering/methods , Nucleotides/chemistry , Amino Acids , Anticodon , Escherichia coli/genetics , Green Fluorescent Proteins/genetics , Microorganisms, Genetically-Modified , Nucleotides/genetics , Recombinant Proteins/genetics
4.
J Am Chem Soc ; 142(5): 2110-2114, 2020 02 05.
Article in English | MEDLINE | ID: mdl-31985216

ABSTRACT

Unnatural base pairs (UBPs) have been developed and used for a variety of in vitro applications as well as for the engineering of semisynthetic organisms (SSOs) that store and retrieve increased information. However, these applications are limited by the availability of methods to rapidly and accurately determine the sequence of unnatural DNA. Here we report the development and application of the MspA nanopore to sequence DNA containing the dTPT3-dNaM UBP. Analysis of two sequence contexts reveals that DNA containing the UBP is replicated with an efficiency and fidelity similar to that of natural DNA and sufficient for use as the basis of an SSO that produces proteins with noncanonical amino acids.


Subject(s)
Base Pairing , Genetic Code , Nanopores , Hydrophobic and Hydrophilic Interactions
5.
J Am Chem Soc ; 141(27): 10644-10653, 2019 07 10.
Article in English | MEDLINE | ID: mdl-31241334

ABSTRACT

Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.


Subject(s)
Nucleotides/genetics , Protein Biosynthesis , Synthetic Biology/methods , Transcription, Genetic , Base Pairing , Deoxyribonucleotides/chemistry , Deoxyribonucleotides/genetics , Genetic Code , Nucleotides/chemistry
6.
Curr Opin Chem Biol ; 46: 196-202, 2018 10.
Article in English | MEDLINE | ID: mdl-30205312

ABSTRACT

Current methods to expand the genetic code enable site-specific incorporation of non-canonical amino acids (ncAAs) into proteins in eukaryotic and prokaryotic cells. However, current methods are limited by the number of codons possible, their orthogonality, and possibly their effects on protein synthesis and folding. An alternative approach relies on unnatural base pairs to create a virtually unlimited number of genuinely new codons that are efficiently translated and highly orthogonal because they direct ncAA incorporation using forces other than the complementary hydrogen bonds employed by their natural counterparts. This review outlines progress and achievements made towards developing a functional unnatural base pair and its use to generate semi-synthetic organisms with an expanded genetic alphabet that serves as the basis of an expanded genetic code.


Subject(s)
Amino Acids/genetics , DNA/genetics , Genetic Code , Genetic Engineering/methods , Amino Acids/chemistry , Animals , Base Pairing , DNA/chemistry , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions
7.
Nanoscale ; 10(21): 10116-10122, 2018 May 31.
Article in English | MEDLINE | ID: mdl-29781024

ABSTRACT

Analysis of single nucleotide variations (SNVs) in DNA and RNA sequences is instrumental in healthcare for the detection of genetic and infectious diseases and drug-resistant pathogens. Here we took advantage of the developments in DNA nanotechnology to design a hybridization sensor, named the 'owl sensor', which produces a fluorescence signal only when it complexes with fully complementary DNA or RNA analytes. The novelty of the owl sensor operation is that the selectivity of analyte recognition is, at least in part, determined by the structural rigidity and stability of the entire DNA nanostructure rather than exclusively by the stability of the analyte-probe duplex, as is the case for conventional hybridization probes. Using two DNA and two RNA analytes we demonstrated that owl sensors differentiate SNVs in a wide temperature range of 5 °C-32 °C, a performance unachievable by conventional hybridization probes including the molecular beacon probe. The owl sensor reliably detects cognate analytes even in the presence of 100 times excess of single base mismatched sequences. The approach, therefore, promises to add to the toolbox for the diagnosis of SNVs at ambient temperatures.


Subject(s)
Base Pair Mismatch , DNA/analysis , Nanostructures , Nucleic Acid Hybridization , DNA Probes , Nucleic Acid Conformation , Nucleotides
8.
J Am Chem Soc ; 140(2): 758-765, 2018 01 17.
Article in English | MEDLINE | ID: mdl-29309130

ABSTRACT

Semisynthetic organisms (SSOs) created from Escherichia coli can replicate a plasmid containing an unnatural base pair (UBP) formed between the synthetic nucleosides dNaM and dTPT3 (dNaM-dTPT3) when the corresponding unnatural triphosphates are imported via expression of a nucleoside triphosphate transporter. The UBP can also be transcribed and used to translate proteins containing unnatural amino acids. However, UBPs are not well retained in all sequences, limiting the information that can be encoded, and are invariably lost upon extended growth. Here we explore the contributions of the E. coli DNA replication and repair machinery to the propagation of DNA containing dNaM-dTPT3 and show that replication by DNA polymerase III, supplemented with the activity of polymerase II and methyl-directed mismatch repair contribute to retention of the UBP and that recombinational repair of stalled forks is responsible for the majority of its loss. This work elucidates fundamental aspects of how bacteria replicate DNA and we use this information to reprogram the replisome of the SSO for increased UBP retention, which then allowed for the first time the construction of SSOs harboring a UBP in their chromosome.


Subject(s)
DNA Replication , Escherichia coli/metabolism , Base Pairing , DNA Repair , Escherichia coli/genetics , Molecular Structure , Plasmids , Synthetic Biology
SELECTION OF CITATIONS
SEARCH DETAIL
...