Molecular Biology Chapter 2 Genetic Material

Chapter II Genetic Material

Molecular nature of genetic material

The genetic material of most organisms is DNA

• The necessary properties of genetic material
  • Stability of physical and chemical properties:
  • be faithfully reproduced and transmitted;
  • some degree of heritable variation;
• How to prove that DNA is genetic material
  • Classic Experiment 1: Diplococcus pneumoniae Transformation Experiment
  • Classic experiment 2: Phage transfection experiment, labeled with sulfur and phosphorus isotopes
  • Classic experiment 3: Chargaff rule,
    • Conclusion 1: DNA base composition is species-specific but not tissue-specific
    • Conclusion 2: A=T, G=C
  • Classic Experiment 4: Double Helix Structure

The genetic material of some organisms is RNA

• The genetic material of some viruses is RNA
• the type of virus;
• RNA virus (HIV, SARS, avian influenza virus):
• Retroviruses/oncogenic RNA viruses:
• Actinomycin D;
• Proviral theory and reverse transcriptase:

Can proteins act as genetic material

• Prion
• The abnormal type will have hereditary properties, and its spatial structure has changed
• 
• The role of PrPsc requires the participation of PrPC
• Misfolded forms of the PrPSc protein can catalyze the conversion of native PrPC
  molecules from the normal soluble Q-helix conformation to the insoluble p
  -sheet conformation, ultimately leading to disease and infection:
• As for whether it can be copied, transcribed, and translated, these have not yet been studied, so whether it is genetic material is still controversial

Section 2 Structure of Nucleic Acids

1 Characteristics of DNA double helix structure

DNA double helix, two strands paired in opposite directions. The thumb is pointing in the upward direction of the spiral, the whole is a right-handed spiral

2 Factors Affecting the Stability of DNA Double Helix Structure

• hydrogen bonding (stabilization)

  • AT: two pairs of hydrogen bonds, GC: three pairs of hydrogen bonds
  • Although the hydrogen bond is a weak interaction, the amount piled up is still relatively large. So DNA with more GC bonds is more stable

• Base stacking power (stable)

  • There are mainly two kinds of weak interactions, the hydrophobic interaction and the van der Waals interaction between molecules.

• Electrostatic force (destruction)

  • There are a large number of negatively charged phosphate groups on the main chain of phosphoribose, and the negative charges repel each other, so DNA should be preserved in saline to neutralize

• Base Molecular Energy (Destruction)

  • When the temperature is high, the internal energy of the DNA molecule will increase, resulting in structural damage

3 Polymorphisms in DNA Structure

Three more important ones, all of which exist in natural organisms and have different meanings

• the difference:

  • Pitch: Fat and thin are not the same, B looks more standard. Slender ones are bigger, chunky ones are smaller
  • Direction: A and B are right-handed helices, Z is left-handed helix (bases are more exposed and more accessible to DNA)
  • B is most cases, A is DNA-RNA, and Z is purine-pyrimidine interval arrangement (Z was originally artificially synthesized, and later found in nature)

• Z has strong immunogenicity and is suitable for eliciting an immune response. Under certain conditions, B can transform into Z type

  

• Since Z exists in nature and will be transformed from B, the meaning of its existence may be:

  • Z-DNA is thermodynamically unstable and prone to unwinding: replication, transcription initiation stages
  • Z-DNA genetic information is exposed on the surface of the helix and is easily recognized by regulatory proteins: so some people speculate that the conversion of B to Z is more related to the regulation of genes

4 DNA multi-strand structure

It is easy to form multiple chains between TC-rich chains and AG-rich chains

H-type DNA, usually the reason we don't know will be attributed to gene regulation, because gene regulation is the thing we don't understand the most

• There is also a four-chain structure
  • Compared with the DNA double helix structure, the thermodynamic and kinetic properties of the G-quadruplex helix
    are very stable.
  • G-DNA-rich sequences are mostly found in some functionally and evolutionarily conserved genomic regions, and the quadruplex structure formed by G-rich DNA strands may be one of the elements for mutual recognition between
    molecules . play some special roles.

5 supercoiled structure of DNA

Covalent, closed, circular CCCDNA (covalently closed circularDNA), and only this will form a supercoiled structure

A left-handed supercoil is a positive supercoil, because normal DNA is a right-handed helix, and a superhelix is ​​superimposed on it with a left-handed helix, so a left-handed supercoil can be understood as a positive

• negative supercoil
• There is a loop, the base will be exposed, which is more important

Negative supercoils are often more important because they can be transformed into DNA with loops, because those with loop structures are very useful

• DNA in aqueous solution tends to be in the B-type state
• The most stable configuration of DNA is 10.5 bp/helix
• Less than 10.5bp/helix develops towards positive supercoil (tightened state)
• More than 10.5bp/helix develops to negative supercoil (relaxed state)
• Almost 5% of the DNA of all organisms is negatively supercoiled (DNA is almost always negatively supercoiled)

Schematic diagram of CCCDNA

If one of the strands is cut by a nuclease, we call it OCDNA (open circle). The broken chain will rotate around the intact one, release the tension, and return to the double helix structure, which is a relatively rigid ring structure

If both are cut, it becomes linear

Therefore, the states of double-stranded circular DNA are supercoiled, circular, and linear.

The first picture on the lower left: the shape is different, the physical and chemical properties are different , and the electrophoresis is different

Second panel from the lower left: Density gradient centrifugation, purification

6 Secondary structure of RNA

• dsRNA: should be A-type double helix structure;
• SSRNA: The local double helix is ​​still A-type structure; others are protrusions and various loop structures;
• Special base pairing: GU forms two pairs of hydrogen bonds;

These can affect stability and functionality

Section 3 Denaturation of Nucleic Acids

1 Denaturation of Nucleic Acids

DNA melting/denaturation

• Under the influence of physical and chemical factors,
  the force to maintain the double helix structure of nucleic acid is destroyed, and the stable double helix structure is loose and irregular single-stranded structure .

• Denaturation leads to changes in the physicochemical properties of nucleic acids, most importantly
  the hyperchromic effect (change in UV absorption) ( $About{D}_{260}$Increase)

• Factors Affecting Degeneration

  • Any factor that destroys
    the force (hydrogen bond, base stacking force) that is conducive to maintaining the DNA double helix structure ,
  • Factors that enhance the forces (electrostatic repulsion, molecular internal energy) that are not conducive to maintaining the DNA double helix structure,
  • Both can promote nucleic acid denaturation.

• Thermal denaturation, the denaturation temperature can reflect the problem

• Melting temperature (Tm)

• What factors affect Tm

  • GC pair content
  • Effect of Salt Ion Concentration
    • When the concentration of salt ions is low, the shielding effect is weakened and Tm is reduced;
    • The concentration of salt ions is high, the hydrophobic interaction is enhanced, and the Tm is increased;

2 Renaturation of Nucleic Acids

DNA annealing/renaturation

• Renaturation : The phenomenon that denatured DNA recovers to form a double helix structure under appropriate conditions is called renaturation.
• Annealing : Heat-denatured DNA can be annealed under slow cooling conditions, which is called annealing (such as the return temperature in PCR).
• Refolding conditions:
  • Two denatured single-stranded DNAs can collide with each other;
  • The two single-stranded DNAs colliding have complementary sequences;
• Refolding factors:
  • Temperature: promotes collision; too high causes denaturation;
  • DNA concentration: the higher the concentration, the higher the chance of collision;
  • DNA sequence complexity: the higher the complexity, the more difficult it is to complement;
• Refolding kinetics

• Start with chemistry

$C_0{t}_{\frac{1}{2}}$It is a very important physical quantity to measure renaturation. It's the same as tm is a measure of degeneration.

• The higher the complexity of the DNA sequence, the longer the renaturation time and the more difficult the renaturation, $C_0{t}_{\frac{1}{2}}$bigger
• So $C_0{t}_{\frac{1}{2}}$can reflect sequence complexity
• can see something

Rapid refolding is generally a sequence with high base repeatability

3 Hybridization of Nucleic Acids

hybridization

• After understanding denaturation and renaturation, we have the concept of hybridization

• The formation of a stable double helix structure between any two single-stranded DNAs or RNAs with complementary paired bases is called nucleic acid hybridization.

• That is, there can be DNA from different origins, forming a double helix

• some methods

1. 
2. Northern Blotting
3. 
4. 
5. Colony HybridizationColony Hybridization