Nucleic Acids [Brazier] Flashcards
Which 2 nucleic acids are purines?
Adenine
Guanine
Which 2 nucleic acids are pyrimidines?
Cytosine
Thymine
NAme the 5 nucleic acids
Adenine Guanine Cytosine Thymine Uracil (instead of thymine in RNA)
What are nucleic acids composed of?
Heterocyclic bases
What is a nucleoside?
A heterocyclic base (nucleic acid) attached to a 5-membered sugar unit
Deoxyadenosine, Deoxyguanosine, Deoxythymidine, Deoxycytidine
What is the sugar unit in DNA?
Deoxyribose
What is the sugar unit in RNA?
Ribose
How are the nucleosides connected?
By phosphate linkages between the 3’-5’ positions
sugar-phosphate backbone
How do nucleic acids form a double helix
Complementary base pairing between 2 strands to form a double helix
A-T, C-G
How do nucleic acids bond?
Via hydrogen bonding
A-T form 2 hydrogen bonds
C-G form 3 hydrogen bonds
What is syn-anti conformation?
The orientation of the heterocyclic base to the sugar
Describe anti conformation
Anti conformation has the smaller H-6 (pyrimidine) or H-8 (purine) above the sugar ring
Describe syn conformation
Syn conformation has the larger O-2 (pyrimidine) or N-3 (purine) above the sugar ring
What is an RNA-DNA helix?
When a strand of RNA folds back on itself to make a double helix
How is an RNA-DNA helix formed?
RNA contains helical regions formed by intramolecular base pairing
When can RNA form a double helix?
During reverse transcription of viral RNA
Transcription of DNA into messenger RNA
Anti-sense therapy
Name 5 drugs that can interact with DNA
Intercalating agents Topoisomerase poisons Alkylating agents Chain cutters Chain terminators
What are intercalating agents?
Molecules which may insert themselves between bases in the DNA helix causes a frameshift mutation during replication
Describe the structure of intercalating agents
They contain heteroaromatic structures to fit between the bases
How do intercalating agents stay in between the DNA bases?
Held in position by Van der Waals interactions
Name 3 intercalating agents
Proflavine - antibacterial
Quinine - anti-malarial
Chloroquinine - anti-malarial
What is Doxorubicin?
A highly effective anti-cancer drug
How does Doxorubicin work?
It approaches the major groove of the DNA and intercalates using its planar tricyclic structure
Its amino group (NH3+) within the sugar can interact with the phosphate backbone
Hinders the normal action of topoisomerase II (enzyme involved in replication) by stabilising the enzyme-DNA complex = DNA strand stays broken
Does not inhibit the enzyme, only hinders, so = poison
What is Topoisomerase II?
An enzyme involved in DNA replication
Catalyses supercoiling
What is the purpose of supercoiling?
Allows the DNA to have a more compact structure so it can fit into the nucleus
How does supercoiling occur?
It requires 1 stretch of the DNA to cross over another
This process is catalysed by topoisomerase II
Name a non-intercalating drug
Fluroquinolones
How do fluoroquinolones work?
The binding site for the drug appears only after DNA has been cleaved by the topoisomerase enzyme
It forms a planar core which stacks through Van der Waals interactions - keeping DNA strands apart & broken
Substituents at C6 and C7 positions can bind to the enzyme
Carbonyl oxygen can form H bonds with DNA
What are alkylating agents?
Highly electrophilic molecules which react with nucleophlic groups (N) within the heterocyclic DNA bases
What type of cross-links can alkylating agents form onto the DNA?
Inter-strand crosslink = disrupts replication and transcription by hindering separation of strands
Intra-strand crosslink = masks DNA structure from enzymes involved in replication and transcription
What is a potential issue with the use of alkylating agents?
They can react with other nucleophilic groups in the body so lack selectivity and often have toxic side effects
Inter strands = cross both strands
Intra strands = crosslinks 2 bases in 1 strand
a
How do nitrogen mustards work as anti-cancer agents?
By forming crosslinks within DNA structures (intra and inter)
How do nitrogen mustards (alkylating agents) react?
SN2 reaction mechanism
Lone pair of the nitrogen attacks the carbon and halogen LG is lost
Very reactive species formed, N has a positive charge
N on nucleotide attacks molecule and breaks the C-N bond
2nd CN bond is formed when N attacks Cl again, and another nucleotide attacks the molecule to break the bond
Crosslink is formed with DNA
How can the toxicity of alkylating agents be reduced?
By ensuring the active species if formed during metabolism
How does Nitrosoureas work as an anti-cancer drug??
It decomposes spontaneously to form an alkylating agent and carbamylating agent
What is a carbamylating agent?
Binds and inactivates proteins
How does Busulfan work as an anti-cancer drug?
Forms inter-strand crosslinks following an Sn2 mechanism
How does Cisplatin work as an anti-cancer drug?
Once it is in the cellular environment, its 2 chloride substituents are displaced by water molecules
The active species then forms intra-strand crosslinks in regions containing adjacent guanine bases
Attachment of the drug disturbs the hydrogen bonding between the heterocyclic bases resulting in local unwinding of the helix
What are ‘chain cutters’?
Drugs that cleave the DNA and prevent DNA ligase (enzyme) from repairing the damage
Works through a radical mechanism resulting in oxidative cleavage of the DNA structure
What are chain terminators?
Molecules which inhibit DNA replication by acting as ‘false substrates’
They lack the functional groups (3’ hydroxyl/OH) required for further chain growth so incorporation into a growing DNA chain = termination
However have 3 phosphate groups attached to build on the back bone (Drug-P-P-P instead of G-P-P-P)
What are the clinical uses of chain terminators?
Used as anti-viral drugs
Target reverse transcriptase in HIV
How can gene transcription be controlled?
By targeting DNA base pairs
Stabilisation of the double helix will inhibit the processes involved in the transcription and ultimately protein synthesis
What can be used to control gene transcription?
Hairpin polyamide structures selectively bind to specific DNA sequences
They must bind in a regulatory region to have any effect on gene expression
Why does RNA provide an attractive drug target
Antisense
siRNA
Why is RNA more difficult to target than DNA?
RNA is more transient
As soon as it is produced it is taken up by the ribosomes to produce proteins
What is an oligonucleotide?
A short nucleic acid polymer which is designed to hybridise specifically to DNA or RNA
Usually made up of 13-25 nucleotides
Can be used in antisense therapy
What is antisense RNA (asRNA)?
A single-stranded RNA which is complementary to a mRNA strand transcribed within the cell
How does an antisense DNA oligonucleotide prevent translation?
Forms a DNA-RNA duplex
The ribosomes cannot process a double-stranded molecule
Briefly describe the process of how DNA leads to the production of a protein
The DNA is transcribed into RNA in the nucleus
The RNA leaves the nucleus and is fed through ribosomes
The ribosomes produce chains of amino acids
These amino acids fold to form proteins
What are the 2 modes of action of antisense therapy?
Steric block = highly stable DNA-RNA duplex formed so ribosome cannot separate it
Cleavage by RNaseH = cleavage of RNA strand of DNA-RNA duplex
How does antisense therapy work?
Blocks translation of single strand of mRNA by causing an antisense DNA oligonucleotide to bind with it
This forms a DNA-RNA duplex which cannot be processed by the ribosomes in order to produce a protein
List the 5 optimal characteristics of antisense therapy
Duplex stability Specificity - target a specific RNA strand Nuclease stability Cellular uptake Toxicity
How can duplex stability be measured?
By UV spectroscopy
A base pair = chromophore
Stacking of the base pairs = decrease in absorbance of the helical structure
How can it be ensured that the antisense oligonucleotide will be stable in physiological environments?
Plot absorbance vs Temperature
Tm (peak) can be used
Body temperature = 37C so it must be stable at this temperature
How can the antisense oligonucleotide be specific?
Due to the well-defined base pairing properties of nucleic acids, the antisense oligonucleotide can be designed to specifically target a particular site
What could be the issues of designing an antisense oligonucleotide specifically to a strand of RNA?
Oligonucleotide too short = could lead to the targeting of other regions of RNA, not specific enough
Oligonucleotide too long = could lead to the targeting of mismatched sequences
What is RNA interference?
Biological process in which miRNA molecules inhibit gene expression, typically causing the destruction of specific mRNA molecules
RNA –> miRNA –> siRNA –> mRNA destroyed
What is micro-RNA?
Small double-stranded RNA molecules (RNA-RNA duplex)
Produced from larger RNA sequences by the Dicer enzyme
miRNA
How does miRNA work?
miRNA produced from larger RNA sequences of by the Dicer enzyme
It then binds to a complex of enzymes known as RISC (RNA inducing silencing complex)
In the RISC, 1 strand is discarded to leaving the rest behind = the small interfering RNA sequence (siRNA), single-stranded
The active RISC recognises a complemetary mRNA, siRNA (still attached to the RISC) binds to it and induces cleavage of the mRNA
Inhibition of protein synthesis
Cleavage of viral RNA halts attack
What is Anti-gene therapy?
Similar to the antisense approach but targets the DNA double helix rather than the RNA
How does Anti-gene therapy work?
Introduction of 3rd strand of DNA added to double helix at the major groove = triple helix
The base pairs in DNA are capable of further hydrogen bonding = Hoogsteen base triplets
List the 2 types of Hoogsteen base triplets which can exist
C+G-C
T+A-T
Explain the C+G-C base triplet
It is more stable but protonation of C leads to a pH dependency
Successive stacking of C+G-C base triplets leads to a destabilisation of the helix (charges stacking)
Explain the T+A-T base triplet
This base triplet does not suffer from the same problems as C+G-C but is inherently less stable
What can be used to measure the stability of the triple helix?
UV spectroscopy
Absorbance vs Temperature
List 2 problems that must be overcome in order for the Anti-gene approach to provide suitable therapeutic action
Delivery - oligonucleotides are highly charged species which will find it difficult to cross the cell membranes
Initial approaches could only target purine bases and not pyrimidines - so a single pyrimidine base in a stretch of purines leads to destabilisation of the helix
